Novel immunoconjugates

ABSTRACT

The present invention generally relates to antigen-specific immunoconjugates for selectively delivering effector moieties that influence cellular activity. More specifically, the invention provides novel immunoconjugates comprising a first antigen binding moiety, an Fc domain and a single effector moiety. In addition, the present invention relates to polynucleotides encoding such immunoconjugates, and vectors and host cells comprising such polynucleotides. The invention further relates to methods for producing the immunoconjugates of the invention, and to methods of using these immunoconjugates in the treatment of disease.

This application is a continuation of U.S. application Ser. No.13/457,039, filed Apr. 26, 2012, which claims benefit under 35 U.S.C.§119 to European Patent Application No. 11164237.7 filed Apr. 29, 2011,the contents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention generally relates to antigen-specificimmunoconjugates for selectively delivering effector moieties thatinfluence cellular activity. In addition, the present invention relatesto polynucleotides encoding such immunoconjugates, and vectors and hostcells comprising such polynucleotides. The invention further relates tomethods for producing the immunoconjugates of the invention, and tomethods of using these immunoconjugates in the treatment of disease.

BACKGROUND

The selective destruction of an individual cell or a specific cell typeis often desirable in a variety of clinical settings. For example, it isa primary goal of cancer therapy to specifically destroy tumor cells,while leaving healthy cells and tissues intact and undamaged. Amultitude of signal transduction pathways in the cell are linked to thecell's survival and/or death. Accordingly, the direct delivery of apathway factor involved in cell survival or death can be used tocontribute to the cell's maintenance or destruction. Similarly, specificfactors may be delivered that stimulate immune effector cells in a tumormicroenvironment, such as natural killer (NK) cells or cytotoxic Tlymphocytes (CTLs), to attack and destroy tumor cells.

Cytokines are cell signaling molecules that participate in regulation ofthe immune system. When used in cancer therapy, cytokines can act asimmunomodulatory agents that have anti-tumor effects and which canincrease the immunogenicity of some types of tumors. However, rapidblood clearance and lack of tumor specificity require systemicadministration of high doses of the cytokine in order to achieve aconcentration of the cytokine at the tumor site sufficient to activatean immune response or have an anti-tumor effect. These high levels ofsystemic cytokine can lead to severe toxicity and adverse reactions.

For use in therapy, it is therefore desirable to specifically deliver asignal transduction pathway factor, such as a cytokine, to a specificsite in vivo (e.g. a tumor or tumor microenvironment in the case ofcancer therapy). This can be achieved by conjugating the factor to atargeting moiety, e.g. an antibody or an antibody fragment, specific forthe site. Early strategies aimed at delivering signal transductionpathway factors, such as cytokines, to a specific site in vivo includedimmunoglobulin heavy chains conjugated to various cytokines, includinglymphotoxin, tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2), andgranulocyte macrophage-colony stimulating factor (GM-CSF) (reviewed e.g.in Lode et al., Pharmacol Ther 80, 277-292 (1998)).

Researchers observed that, not only were they able to target cytokinesto specific sites in vivo, they were also able to take advantage of thefact that monoclonal antibodies have longer serum half-lives than mostother proteins. Given the systemic toxicity associated with high dosesof certain unconjugated cytokines, e.g. IL-2, the ability of animmunoglobulin-cytokine fusion protein to maximize therapeuticallybeneficial biological activities at a desired site, e.g. in a tumor,whilst keeping systemic side effects to a minimum at a lower dose ledresearchers to believe that immunoglobulin-cytokine immunoconjugateswere optimal therapeutic agents.

Nevertheless, there are certain disadvantages associated with theimmunoglobulin-cytokine immunoconjugtates known in the art. For example,these immunoconjugates have at least one cytokine coupled to each of thetwo immunoglobulin heavy chains, resulting in an immunoconjugate withbivalent target binding and two or more cytokine moieties (reviewed e.g.in Chang et al., Expert Opin Drug Discovery 4, 181-194 (2009), orOrtiz-Sanchez et al., Expert Opin Biol Ther 8, 609-632 (2008)). FIG. 1depicts a conventional immunoglobulin-cytokine immunoconjugate as it isknown in the art, where a cytokine is fused to the C-terminus of each ofthe two antibody heavy chains. Due to the presence of two or morecytokine moieties, such an immunoconjugate has a high avidity to therespective cytokine receptor (for example, picomolar affinity in thecase of IL-2), and thus is targeted rather to the immune effector cellsexpressing the cytokine receptor than to the target antigen of theimmunoglobulin (nM affinity) to which the cytokine is linked. Moreover,conventional immunoconjugates are known to be associated with infusionreactions (see e.g. King et al., J Clin Oncol 22, 4463-4473 (2004)),resulting at least partially from activation of cytokine receptors onimmune effector cells in peripheral blood by the immunoconjugate'scytokine moieties.

Additionally, via their Fc domain, immunoglobulin-cytokineimmunoconjugates can activate complement and interact with Fc receptors.This inherent immunoglobulin feature has been viewed unfavorably becausetherapeutic immunoconjugates may be targeted to cells expressing Fcreceptors rather than the preferred antigen-bearing cells. Moreover, thesimultaneous activation of cytokine receptors and Fc receptor signalingpathways leading to cytokine release, especially in combination with thelong half-life of immunoglobulin fusion proteins, make their applicationin a therapeutic setting difficult due to systemic toxicity.

One approach to overcoming this problem is the use of immunoglobulinfragments devoid of an Fc domain, such as scFv or Fab fragments, inimmunoconjugates. Examples of immunoglobulin fragment-cytokineimmunoconjugates include the scFv-IL-2 immunoconjugate as set forth inPCT publication WO 2001/062298, the scFv-IL-12-scFv immunoconjugate asset forth in PCT publication WO 2006/119897 (wherein each of the twoscFv fragments is connected to a subunit of the IL-12 heterodimer thatis held together by disulfide bond(s)) or the Fab-IL-2-Fabimmunoconjugates as set forth in PCT publication WO 2011/020783. Boththe tumor-binding reactivity of the immunoglobulin parent molecule andthe functional activity of the cytokine are maintained in most of thesetypes of immunoconjugates, however the half-life of such constructs isconsiderably shorter than of immunoglobulin fusion proteins.

Therefore there remains a need for immunoconjugates with improvedproperties, for greater therapeutic effectiveness and a reduction in thenumber and severity of the side effects of these products (e.g.,toxicity, destruction of non-tumor cells, etc.).

The present invention provides immunoglobulin-like immunoconjugates thatexhibit improved efficacy, high specificity of action, reduced toxicity,and improved half-life and stability in blood relative to knownimmunoconjugates.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the inventors' recognitionthat immunoconjugates comprising more than one effector moiety, such ase.g. a cytokine, may be targeted to the respective effector moietyreceptor rather than the target antigen of the antigen binding moiety ofthe immunoconjugate. Therefore, in one aspect the invention provides animmunoconjugate comprising a first antigen binding moiety, an Fc domainconsisting of two subunits, and an effector moiety, wherein not morethan one effector moiety is present. In one embodiment the effectormoiety is fused to the amino- or carboxy-terminal amino acid of one ofthe two subunits of the Fc domain, optionally through a linker peptide.In one embodiment the first antigen binding moiety is fused to theamino-terminal amino acid of one of the two subunits of the Fc domain,optionally through a linker peptide or an immunoglobulin hinge region.

In one embodiment the first antigen binding moiety comprises an antigenbinding domain of an antibody. In a particular embodiment the firstantigen binding moiety is a Fab molecule. In certain embodiments the Fcdomain comprises a modification promoting heterodimerization of twonon-identical polypeptide chains. In a specific embodiment saidmodification is a knob-into-hole modification, comprising a knobmodification in one of the subunits of the Fc domain and a holemodification in the other one of the two subunits of the Fc domain. In aparticular embodiment the effector moiety is fused to the amino- orcarboxy-terminal amino acid of the subunit of the Fc domain comprisingthe knob modification.

In one embodiment the Fc domain is an IgG Fc domain, particularly anIgG₁ Fc domain. In a particular embodiment the Fc domain is human.

In certain embodiments of the invention the Fc domain is engineered tohave altered binding to an Fc receptor, specifically altered binding toan Fcγ receptor, and/or altered effector function, specifically alteredantibody-dependent cell-mediated cytotoxicity (ADCC).

Although the presence of an Fc domain is essential for prolonging thehalf-life of the immunoconjugate, the inventors realize that in somesituations it will be beneficial to eliminate effector functionsassociated with engagement of Fc receptors by the Fc domain. Hence, inparticular embodiments the altered binding to an Fc receptor and/oreffector function is reduced binding and/or effector function. In aspecific such embodiment the Fc domain comprises one or more amino acidmutation that reduces the binding of the Fc domain to an Fc receptor,particularly an Fcγ receptor. Preferably, such an amino acid mutationdoes not reduce binding to FcRn receptors. In one embodiment the Fcdomain comprises an amino acid substitution at position P329. In aparticular embodiment the Fc domain comprises the amino acidsubstitutions L234A, L235A and P329G in each of its subunits.

On the other hand, there may be situations where it is desirable toenhance the effector functions of immunoconjugates. Hence, in certainembodiments the Fc domain of the immunoconjugate of the invention isengineered to have altered binding to an Fc receptor, specifically anFcγ receptor, more specifically an FcγIIIa receptor, and/or alteredeffector function, wherein the altered binding and/or effector functionis increased binding and/or effector function. In one such embodimentthe Fc domain is engineered to have an altered oligosaccharidestructure, as compared to a non-engineered Fc domain. In a particularsuch embodiment the Fc domain comprises an increased proportion ofnon-fucosylated oligosaccharides, as compared to a non-engineered Fcdomain. In a more specific embodiment the Fc domain comprises at least20%, particularly at least 50%, more particularly at least 70%non-fucosylated oligosaccharides. In another specific embodiment the Fcdomain comprises an increased proportion of bisected oligosaccharides,as compared to a non-engineered Fc domain. In yet another specificembodiment the Fc domain comprises an increased proportion of bisected,non-fucosylated oligosaccharides, compared to a non-engineered Fcdomain. In some embodiments said altered oligosaccharide structureresults from increased β(1,4)-N-acetylglucosaminyltransferase III(GnTIII) activity in a host cell used for expression of theimmunoconjugate.

In a particular aspect, the invention provides immunoconjugates thatcomprise a first and a second antigen binding moiety, an Fc domainconsisting of two subunits, and an effector moiety, wherein not morethan one effector moiety is present. In one embodiment the first and thesecond antigen binding moiety and the Fc domain are part of animmunoglobulin molecule. In certain embodiments the immunoconjugateessentially consists of an immunoglobulin molecule and an effectormoiety and optionally one or more linker sequences. In a particularembodiment the immunoglobulin molecule is an IgG class immunoglobulin.In an even more particular embodiment the immunoglobulin is an IgG₁subclass immunoglobulin. In one embodiment the effector moiety is fusedto the carboxy-terminal amino acid of one of the immunoglobulin heavychains, optionally through a linker peptide.

In a particular embodiment the immunconjugate of the invention comprisesan immunoglobulin molecule comprising two antigen binding moieties andan Fc domain, and an effector moiety fused to the carboxy-terminal aminoacid of one of the immunoglobulin heavy chains, wherein not more thanone effector moiety is present and wherein the Fc domain is engineeredto have reduced binding to an Fc receptor, specifically altered bindingto an Fcγ receptor, and/or reduced effector function.

In certain embodiments said first antigen binding moiety, or said firstand said second antigen binding moiety, is directed to an antigenassociated with a pathological condition, such as an antigen presentedon a tumor cell or in a tumor cell environment, at a site ofinflammation, or on a virus-infected cell. In a more specific embodimentsaid antigen is selected from the group of Fibroblast Activation Protein(FAP), the A1 domain of Tenascin-C (TNC A1), the A2 domain of Tenascin-C(TNC A2), the Extra Domain B of Fibronectin (EDB), CarcinoembryonicAntigen (CEA), and Melanoma-associated Chondroitin Sulfate Proteoglycan(MCSP).

In certain embodiments the effector moiety is a single chain effectormoiety. In a particular embodiment the effector moiety is a cytokine. Inone embodiment said cytokine is selected from the group of IL-2, IL-7,IL-10, IL-12, IL-15, IFN-α and IFN-γ. In a particular embodiment saidcytokine is IL-2. In an even more particular embodiment said cytokine isa mutant IL-2 polypeptide having reduced binding affinity to theα-subunit of the IL-2 receptor. In a specific embodiment said mutantIL-2 polypeptide comprises an amino acid substitution at one or morepositions selected from the positions corresponding to residues 42, 45and 72 of human IL-2. In another particular embodiment the cytokine isIL-10. In yet another embodiment, the cytokine is IL-15, particularly amutant IL-15 polypeptide having reduced binding affinity to theα-subunit of the IL-15 receptor. In another embodiment, the cytokine isIFN-α.

According to another aspect of the invention there is provided anisolated polynucleotide encoding an immunoconjugate of the invention ora fragment thereof. The invention further provides an expression vectorcomprising the isolated polynucleotide of the invention, and a host cellcomprising the isolated polynucleotide or the expression vector of theinvention. In some embodiments the host cell is a eukaryotic cell,particularly a mammalian cell. In some embodiments, the host cell hasbeen manipulated to express increased levels of one or more polypeptideshaving β(1,4)-N-acetylglucosaminyltransferase III (GnTIII) activity. Inone such embodiment the host cell has been further manipulated toexpress increased levels of one or more polypeptides havingα-mannosidase II (ManII) activity.

In another aspect is provided a method of producing the immunoconjugatesof the invention, comprising the steps of a) culturing the host cell ofthe invention under conditions suitable for the expression of theimmunoconjugate and b) recovering the immunoconjugate. The inventionalso encompasses an immunoconjugate produced by the method of theinvention.

The invention further provides a pharmaceutical composition comprisingan immunoconjugate of the invention and a pharmaceutically acceptablecarrier.

Also encompassed by the invention are methods of using theimmunoconjugates and pharmaceutical compositions of the invention. Inone aspect the invention provides an immunoconjugate or a pharmaceuticalcomposition of the invention for use as a medicament. In one aspect isprovided an immunoconjugate or a pharmaceutical composition according tothe invention for use in the treatment of a disease in an individual inneed thereof. In a specific embodiment the disease is cancer. In otherembodiments the disease is an inflammatory disorder. In a particularsuch embodiment the immunoconjugate comprises an IL-10 effector moiety.

Also provided is the use of an immunoconjugate of the invention for themanufacture of a medicament for the treatment of a disease in anindividual in need thereof as well as a method of treating a disease inan individual, comprising administering to said individual atherapeutically effective amount of a composition comprising theimmunoconjugate according to the invention in a pharmaceuticallyacceptable form. In a specific embodiment the disease is cancer. Inother embodiments the disease is an inflammatory disorder. In aparticular such embodiment the immunoconjugate comprises an IL-10effector moiety.

In any of the above embodiments the individual preferably is a mammal,particularly a human.

In a further aspect, the invention provides a conjugate comprising afirst Fab molecule which does not specifically bind any antigen, an Fcdomain consisting of two subunits, and an effector moiety, wherein notmore than one effector moiety is present. In a particular embodiment thefirst Fab molecule comprises the heavy chain variable region sequence ofSEQ ID NO: 299 and the light chain variable region sequence of SEQ IDNO: 297. In one embodiment, the effector moiety is fused to the amino-or carboxy-terminal amino acid of one of the two subunits of the Fcdomain, optionally through a linker peptide. In another embodiment, thefirst Fab molecule is fused to the amino-terminal amino acid of one ofsaid two subunits of the Fc domain, optionally through a linker peptideor an immunoglobulin hinge region. In one embodiment, the conjugatecomprises (i) an immunoglobulin molecule, comprising a first and asecond Fab molecule which do not specifically bind any antigen and an Fcdomain, and (ii) an effector moiety, wherein not more than one effectormoiety is present. In one embodiment the immunoglobulin molecule is anIgG class immunoglobulin, particularly an IgG1 subclass immunoglobulin.In a particular embodiment the immunoglobulin molecule comprises theheavy chain variable region sequence of SEQ ID NO: 299 and the lightchain variable region sequence of SEQ ID NO: 297.

Specifically, the heavy chain variable region sequence of SEQ ID NO: 299and the light chain variable region sequence of SEQ ID NO: 297 arecomprised in the first and the second Fab molecule of the immunoglobulinmolecule. In one embodiment, the effector moiety is fused to thecarboxy-terminal amino acid of one of the immunoglobulin heavy chains,optionally through a linker peptide.

In certain embodiments the Fc domain of the conjugate comprises amodification promoting heterodimerization of the non-identicalpolypeptide chains. In a specific embodiment, said modification is aknob-into-hole modification, comprising a knob modification in one ofthe subunits of the Fc domain and a hole modification in the other oneof the two subunits of the Fc domain. In a particular embodiment, theeffector moiety is fused to the amino- or carboxy-terminal amino acid ofthe subunit of the Fc domain comprising the knob modification. In oneembodiment, the Fc domain is an IgG Fc domain, particularly an IgG₁ Fcdomain. In a particular embodiment, the Fc domain is human. In someembodiments, the Fc domain is engineered to have altered binding to anFc receptor, specifically altered binding to an Fcγ receptor, and/oraltered effector function, specifically altered ADCC. In someembodiments the Fc domain of the conjugate is engineered to have reducedbinding to an Fc receptor, specifically reduced binding to an Fcγreceptor, and/or reduced effector function, specifically reduced ADCC.In one embodiment, the Fc domain comprises one or more amino acidmutation that reduces the binding of the Fc domain to an Fc receptor,particularly an Fcγ receptor. In a specific embodiment the amino acidmutation is an amino acid substitution at position P329. In a particularembodiment, the Fc domain of the conjugate comprises the amino acidsubstitutions L234A, L235A and P329G in each of its subunits. In anotherembodiment of the conjugate of the invention, the Fc domain isengineered to have altered binding to an Fc receptor and/or alteredeffector function, wherein said altered binding and/or effector functionis increased binding and/or effector function. In one embodiment of theconjugate of the invention, the Fc domain is engineered to have analtered oligosaccharide structure, as compared to a non-engineered Fcdomain. In a specific embodiment, the Fc domain described abovecomprises an increased proportion of non-fucosylated oligosaccharides,as compared to a non-engineered Fc domain.

In a further embodiment of the conjugate of the invention, the conjugatecomprises a first and a second Fab molecule. In one embodiment, thefirst and the second Fab molecule each comprises the heavy chainvariable region sequence of SEQ ID NO: 299 and the light chain variableregion sequence of SEQ ID NO: 297. In one embodiment, the first and saidsecond Fab molecule and said Fc domain are part of an immunoglobulinmolecule. In a particular embodiment, the immunoglobulin molecule is anIgG class immunoglobulin. In an even more particular embodiment, theimmunoglobulin molecule is an IgG₁ subclass immunoglobulin. In oneembodiment, the effector moiety is fused to the carboxy-terminal aminoacid of one of the immunoglobulin heavy chains, optionally through alinker peptide.

In some embodiments of the conjugate of the invention, said effectormoiety is a single chain effector moiety. In one embodiment the effectormoiety is a cytokine, particularly IL-2. In another embodiment, saidcytokine is a mutant IL-2 polypeptide having reduced binding affinity tothe α-subunit of the IL-2 receptor. In a specific embodiment, saidmutant IL-2 polypeptide comprises an amino acid substitution at one ormore positions selected from the positions corresponding to residues 42,45 and 72 of human IL-2.

Additionally, the conjugate can incorporate, alone or in combination,any of the features described herein in relation to the formats, the Fcdomain or the effector moiety of the immunoconjugates of the invention.

The invention also provides an isolated polynucleotide encoding theconjugate of the invention of a fragment thereof, as described above. Ina specific embodiment, the isolated polynucleotide comprises a sequencethat is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to the sequence of SEQ ID NO: 298 or SEQ ID NO: 300. Theinvention further provides an expression vector comprising the isolatedpolynucleotide, and a host cell comprising the isolated polynucleotideor the expression vector of the invention. In another aspect is provideda method of producing the conjugate of the invention described above,comprising the steps of a) culturing the host cell of the inventionunder conditions suitable for the expression of the conjugate and b)recovering the conjugate. The invention also encompasses a conjugate,described above, produced by the method of the invention, comprising thesteps of a) culturing the host cell of the invention under conditionssuitable for the expression of the conjugate and b) recovering theconjugate.

The invention further provides a pharmaceutical composition comprisingthe conjugate of the invention described above and a pharmaceuticallyacceptable carrier. Furthermore, the conjugate can be employed in themethods of use described herein for the immunoconjugates of theinvention. In one embodiment, the conjugate as described above, or thepharmaceutical composition described above, is for use in the treatmentof a disease in an individual in need thereof or for the manufacture ofa medicament for the treatment of a disease in an individual in needthereof.

In a further aspect of the invention, a method of treating a disease inan individual is provided, comprising administering to said individual atherapeutically effective amount of a composition comprising theconjugate of the invention as described above, in a pharmaceuticallyacceptable form.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of typical immunoglobulin-cytokineimmunoconjugate as known in the art, with a cytokine (dotted) fused tothe C-terminus of each of the two immunoglobulin heavy chains.

FIGS. 2A-C. Schematic representation of novel immunoconjugates accordingto the invention, comprising not more than one effector moiety (dotted).The effector moiety is fused, optionally via a linker peptide (greyboxes) to the carboxy-terminal (FIGURES A and B) or the amino-terminalamino acid (FIGURE C) of the Fc domain. The immunoconjugate comprisesone (FIGURES B and C) or more (typically two, FIGURE A) antigen bindingmoieties, which may be Fab fragments comprising antibody heavy and lightchain variable domains (hatched). The Fc domain may comprise amodification promoting heterodimerization of two non-identicalpolypeptide chains (black dot) and/or a modification altering Fcreceptor binding and/or effector function (black star).

FIGS. 3A-D. Purification of FAP-targeted 4G8-based IgG-IL-2 quadruplemutant (qm) immunoconjugate. FIG. 3A) Elution profile of the Protein Aaffinity chromatography step. FIG. 3B) Elution profile of the sizeexclusion chromatography step. FIG. 3C) Analytical SDS-PAGE (NuPAGENovex Bis-Tris Mini Gel, Invitrogen, MOPS running buffer) of the finalproduct. FIG. 3D) Analytical size exclusion chromatography of the finalproduct on a Superdex 200 column (97% monomer content).

FIGS. 4A-D. Purification of FAP-targeted 28H1-based IgG-IL-2 qmimmunoconjugate. FIG. 4A) Elution profile of the Protein A affinitychromatography step. FIG. 4B) Elution profile of the size exclusionchromatography step. FIG. 4C) Analytical SDS-PAGE (reduced: NuPAGE NovexBis-Tris Mini Gel, Invitrogen, MOPS running buffer; non-reduced: NuPAGETris-Acetate, Invitrogen, Tris-Acetate running buffer) of the finalproduct. FIG. 4D) Analytical size exclusion chromatography of the finalproduct on a Superdex 200 column (100% monomer content).

FIGS. 5A-D. Purification of FAP-targeted 28H1-based IgG-IL-2 qmimmunoconjugate from CHO cells. FIG. 5A) Elution profile of the ProteinA affinity chromatography step. FIG. 5B) Elution profile of the sizeexclusion chromatography step. FIG. 5C) Analytical SDS-PAGE (NuPAGENovex Bis-Tris Mini Gel, Invitrogen, MOPS running buffer) of the finalproduct. FIG. 5D) Analytical size exclusion chromatography of the finalproduct on a Superdex 200 column (100% monomer content).

FIGS. 6A-D. Purification of FAP-targeted 4B9-based IgG-IL-2 qmimmunoconjugate. FIG. 6A) Elution profile of the Protein A affinitychromatography step. FIG. 6B) Elution profile of the size exclusionchromatography step. FIG. 6C) Analytical SDS-PAGE (NuPAGE Novex Bis-TrisMini Gel, Invitrogen, MOPS running buffer) of the final product. FIG.6D) Analytical size exclusion chromatography of the final product on aSuperdex 200 column (100% monomer content).

FIGS. 7A-D. Purification of CEA-targeted CH1A1A 98/99 2F1-based IgG-IL-2qm immunoconjugate. FIG. 7A) Elution profile of the Protein A affinitychromatography step. FIG. 7B) Elution profile of the size exclusionchromatography step. FIG. 7C) Analytical capillary electrophoresis SDS(Caliper) of the final product. FIG. 7D) Analytical size exclusionchromatography of the final product on a TSKgel G3000 SW XL column(98.8% monomer content).

FIGS. 8A-D. Purification of TNC A2-targeted 2B10-based IgG-IL-2 qmimmunoconjugate. FIG. 8A) Elution profile of the Protein A affinitychromatography step. FIG. 8B) Elution profile of the size exclusionchromatography step. FIG. 8C) Analytical capillary electrophoresis SDS(Caliper) of the final product. FIG. 8D) Analytical size exclusionchromatography of the final product on a TSKgel G3000 SW XL column (100%monomer content).

FIGS. 9A-D. Purification of untargeted DP47GS-based IgG-IL-2 qmimmunoconjugate. FIG. 9A) Elution profile of the Protein A affinitychromatography step. FIG. 9B) Elution profile of the size exclusionchromatography step. FIG. 9C) Analytical SDS-PAGE (NuPAGE Novex Bis-TrisMini Gel, Invitrogen, MOPS running buffer) of the final product. FIG.9D) Analytical size exclusion chromatography of the final product on aSuperdex 200 column (100% monomer content).

FIG. 10. Binding of FAP-targeted 4G8-based IgG-IL-2 qm immunoconjugateto human FAP expressed on stably transfected HEK 293 cells as measuredby FACS, compared to the corresponding Fab-IL-2 qm-Fab construct.

FIG. 11. Interferon (IFN)-γ release on NK92 cells induced byFAP-targeted 4G8-based IgG-IL-2 qm immunoconjugate in solution, comparedto the 28H1-based Fab-IL-2 qm-Fab construct.

FIGS. 12A-D. Detection of phosphorylated STATS by FACS in different celltypes after stimulation for 20 min with FAP-targeted 4G8-based IgG-IL-2qm immunoconjugate in solution, compared to the 28H1-based Fab-IL-2-Faband Fab-IL-2 qm-Fab constructs as well as Proleukin. FIG. 12A) NK cells(CD3⁻CD56⁺); FIG. 12B) CD8⁺ T cells (CD3⁺CD8⁺); FIG. 12C) CD4⁺ T cells(CD3⁺CD4⁺CD25⁻CD127⁺); FIG. 12D) regulatory T cells (CD4⁺CD25⁺FOXP3⁺).

FIG. 13. Binding of TNC A2-targeted 2B10 IgG-IL-2 qm and correspondingunconjugated IgG to TNC A2-expressing U87MG cells, as measured by FACS.

FIG. 14. Induction of NK92 cell proliferation by TNC A2-targeted 2B10IgG-IL-2 qm, CEA-targeted CH1A1A 98/99 2F1 IgG-IL-2 qm and CH1A1A 98/992F1 IgG-IL-2 wt immunoconjugates.

FIG. 15. Induction of NK92 cell proliferation by FAP-targeted 4B9IgG-IL-2 qm and 4B9 IgG-IL-2 wt immunoconjugates.

FIG. 16. Killing (as measured by LDH release) of CEA-overexpressing A549tumor cells by PBMCs through ADCC mediated by glycoengineered (ge) andwildtype (wt) CH1A1A IgG-IL-2 qm immunoconjugates, compared tounconjugated glycoengineered CH1A1A IgG.

FIGS. 17A-D. Purification of untargeted DP47GS IgG-IL-2 wtimmunoconjugate. FIG. 17A) Elution profile of the Protein A affinitychromatography step. FIG. 17B) Elution profile of the size exclusionchromatography step. FIG. 17C) Analytical SDS-PAGE (NuPAGE NovexBis-Tris Mini Gel, Invitrogen, MOPS running buffer) of the finalproduct. FIG. 17D) Analytical size exclusion chromatography of the finalproduct on a Superdex 200 column (99.6% monomer content).

FIGS. 18A-D. Purification of 28H1-based FAP-targeted 28H1 IgG-IL-2 wtimmunoconjugate. FIG. 18A) Elution profile of the Protein A affinitychromatography step. FIG. 18B) Elution profile of the size exclusionchromatography step. FIG. 18C) Analytical SDS-PAGE (NuPAGE NovexBis-Tris Mini Gel, Invitrogen, MOPS running buffer) of the finalproduct. FIG. 18D) Analytical size exclusion chromatography of the finalproduct on a Superdex 200 column (99.6% monomer content).

FIGS. 19A-D. Purification of CEA-targeted CH1A1A 98/99 2F1-basedIgG-IL-2 wt immunoconjugate. FIG. 19A) Elution profile of the Protein Aaffinity chromatography step. FIG. 19B) Elution profile of the sizeexclusion chromatography step. FIG. 19C) Analytical capillaryelectrophoresis SDS (Caliper) of the final product. FIG. 19D) Analyticalsize exclusion chromatography of the final product on a TSKgel G3000 SWXL column (100% monomer content).

FIGS. 20A-D. Purification of FAP-targeted 4B9-based IgG-IL-2 wtimmunoconjugate. FIG. 20A) Elution profile of the combined Protein Aaffinity and size exclusion chromatography. FIG. 20B) Zoom on theelution profile of the size exclusion chromatography step in A. FIG.20C) Analytical SDS-PAGE (NuPAGE Novex Bis-Tris Mini Gel, Invitrogen,MOPS running buffer) of the final product. FIG. 20D) Analytical sizeexclusion chromatography of the final product on a TSKgel G3000 SW XLcolumn (98.5% monomer content).

FIGS. 21A-C. FIG. 21A) Analytical SDS-PAGE (NuPAGE Novex Bis-Tris MiniGel (Invitrogen), NuPAGE LDS sample buffer (4×), heated for 10 min at70° C., MOPS buffer, 160 V, 60 min, MW marker Mark 12, unstainedstandard (Invitrogen, M) of reduced (1) and non-reduced (2) 2B10IgG-IL-10M1. FIG. 21B) SPR-based affinity determination (ProteOn XPR36)of 2B10 IgG-IL-10M1 to human TNC A2 fitted globally to a 1:1 interactionmodel. (chip: NLC; ligand: TNCA2 (250 RU); analyte: TNCA2 2B10IgG-IL-10M1 164 kDa; concentration range analyte: 50, 10, 2, 0.4, 0.08,0 nM; association time: 180 s; dissociation time: 600 s; flow rate: 50μl/min; k_(on) 1.80×10⁶ l/Ms; k_(off): 9.35×10⁻⁵ l/s; K_(D): 52 pM).FIG. 21C) SPR-based affinity determination (ProteOn XPR36) of 2B10IgG-IL-10M1 to human IL-10R1 fitted globally to a 1:1 interaction model(chip: NLC; ligand: IL-10R1 (1600RU); analyte: TNCA2 2B10 IgG-IL-10M1164 kDa; concentration range analyte: 50, 10, 2, 0.4, 0.08, 0 nM;association time: 180 s; dissociation time: 600 s; flow rate: 50 μl/min;k_(on) 5.56×10⁵ l/Ms; k_(off): 2.89×10⁻⁴ l/s; K_(D): 520 pM).

FIGS. 22A-C. FIG. 22A) Analytical SDS-PAGE (NuPAGE Novex Bis-Tris MiniGel (Invitrogen), NuPAGE LDS sample buffer (4×), heated for 10 min at70° C., MOPS buffer, 160 V, 60 min, MW marker Mark 12, unstainedstandard (Invitrogen, M) of reduced (1) and non-reduced (2) 4G8IgG-IL-10M1. FIG. 22B) SPR-based affinity determination (ProteOn XPR36)of 4G8 IgG-IL-10M1 to human FAP fitted globally to a 1:1 interactionmodel (chip: GLM; ligand: huFAP (500RU); analyte: FAP 4G8 IgG-IL-10M1164 kDa; concentration range analyte: 10, 2, 0.4, 0.08, 0 nM;association time: 180 s; dissociation time: 600 s; flow rate: 50 μl/min;k_(on) 6.68×10⁵ l/Ms; k_(off): 1.75×10⁻⁵ l/s; K_(D): 26 pM). FIG. 22C)SPR-based affinity determination (ProteOn XPR36) of 4G8 IgG-IL-10M1 tohuman IL-10R1 fitted globally to a 1:1 interaction model (chip: NLC;ligand: IL 10R1 (1600RU); analyte: FAP 4G8 IgG-IL-10M1 164 kDa;concentration range analyte: 50, 10, 2, 0.4, 0.08, 0 nM; associationtime: 180 s; dissociation time: 600 s; flow rate: 50 μl/min; k_(on):3.64×10⁵ l/Ms; k_(off): 2.96×10⁻⁴ l/s; K_(D): 815 pM).

FIGS. 23A-C. Purification of FAP-targeted 4B9-based “1+1” IgG-IL-2 qmimmunoconjugate. FIG. 23A) Elution profile of the combined Protein Aaffinity and size exclusion chromatography. FIG. 23B) AnalyticalSDS-PAGE (NuPAGE Novex Bis-Tris Mini Gel, Invitrogen, MOPS runningbuffer) of the final product. FIG. 23C) Analytical size exclusionchromatography of the final product on a TSKgel G3000 SW XL column(99.2% monomer content).

FIGS. 24A-C. Purification of FAP-targeted 28H1-based “1+1” IgG-IL-2 qmimmunoconjugate. FIG. 24A) Elution profile of the combined Protein Aaffinity and size exclusion chromatography. FIG. 24B) AnalyticalSDS-PAGE (NuPAGE Novex Bis-Tris Mini Gel, Invitrogen, MOPS runningbuffer) of the final product. FIG. 24C) Analytical size exclusionchromatography of the final product on a TSKgel G3000 SW XL column (100%monomer content).

FIGS. 25A-C. Purification of FAP-targeted 4B9-based “1+1” IgG-IL-7immunoconjugate. FIG. 25A) Elution profile of the combined Protein Aaffinity and size exclusion chromatography. FIG. 25B) Analyticalcapillary electrophoresis SDS (Caliper) of the final product. FIG. 25C)Analytical size exclusion chromatography of the final product on aTSKgel G3000 SW XL column (98.6% monomer content).

FIGS. 26A-D. Purification of FAP-targeted 4B9-based “1+1” IgG-IFN-αimmunoconjugate. FIG. 26A) Elution profile of the Protein A affinitychromatography step. FIG. 26B) Elution profile of the size exclusionchromatography step. FIG. 26C) Analytical capillary electrophoresis SDS(Caliper) of the final product. FIG. 26D) Analytical size exclusionchromatography of the final product on a TSKgel G3000 SW XL column(92.8% monomer content).

FIG. 27. Induction of NK92 cell proliferation by FAP-targeted 4B9 “1+1”IgG-IL-2 qm and 28H1 “1+1” IgG-IL-2 wt immunoconjugates, compared tocorresponding IgG-IL-2 constructs.

FIGS. 28A-B. Proliferation of PHA-activated (FIG. 28A) CD4 and (FIG.28B) CD8 T cells induced by 4B9 “1+1” IgG-IL-7 and 4B9 “1+1” IgG-IL-2 qmimmunoconjugates, compared to IgG-IL-2 qm and IgG-IL-2 wt constructs.

FIG. 29. Induction of Daudi cell proliferation by 4B9 “1+1” IgG-IFN-α,compared to Roferon A.

FIGS. 30A-B. Serum concentrations of IL-2 immunoconjugates after asingle i.v. administration of FAP-targeted (FIG. 30A) and untargeted(FIG. 30B) IgG-IL-2 constructs comprising either wild-type (wt) orquadruple mutant (qm) IL-2.

FIG. 31. Tissue distribution of FAP-targeted 28H1 IgG-IL qm compared tounconjugated FAP-targeted 28H1 IgG and 4B9 IgG, as well as untargetedDP47GS IgG, 24 hours after i.v. injection.

FIG. 32. Binding of 28H1 IgG-IL-2 qm and 28H1 IgG-(IL-2 qm)₂immunoconjugates to NK92 cells as determined by FACS.

FIGS. 33A-C. Proliferation of NK cells upon incubation with differentFAP-targeted 28H1 IL-2 immunoconjugates or Proleukin for 4 (FIG. 33A), 5(FIG. 33B) or 6 (FIG. 33C) days.

FIGS. 34A-C. Proliferation of CD4 T-cells upon incubation with differentFAP-targeted 28H1 IL-2 immunoconjugates or Proleukin for 4 (FIG. 34A), 5(FIG. 34B) or 6 (FIG. 34C) days.

FIGS. 35A-C. Proliferation of CD8 T-cells upon incubation with differentFAP-targeted 28H1 IL-2 immunoconjugates or Proleukin for 4 (FIG. 35A), 5(FIG. 35B) or 6 (FIG. 35C) days.

FIGS. 36A-B. Proliferation of pre-activated CD8 (FIG. 36A) and CD4 (FIG.36B) T cells after six days incubation with different IL-2immunoconjugates.

FIG. 37. Activation induced cell death of CD3⁺ T cells after six daysincubation with different IL-2 immunoconjugates and overnight treatmentwith anti-Fas antibody.

FIGS. 38A-B. Serum concentrations of IL-2 immunoconjugates after asingle i.v. administration of untargeted DP47GS IgG-IL-2 constructscomprising either wild-type (FIG. 38A) or quadruple mutant IL-2 (FIG.38B).

FIG. 39. Binding of DP47GS IgG to different antigens. Binding wasdetected in an ELISA-based assay with the antigens captured on theplate. A human IgG1 antibody which exhibits unspecific binding to almostall of the captured antigens was used as positive control, blank samplesdid not contain any antibody.

FIGS. 40A-L. Binding of DP47GS IgG with or without LALA P329G mutationin the Fc domain to subsets of fresh (FIGS. 40A-D), PHA-L activated(FIGS. 40E-H) and re-stimulated (FIGS. 40I-L) human PBMCs, as determinedby FACS analysis. Upper left panel: B cells (in FIG. 40A and FIG. 40E)or CD4⁺ T cells (in FIG. 40I); upper right panel: CD8⁺ T cells (in FIG.40B, FIG. 40F and FIG. 40J); lower left panel (in FIG. 40C, FIG. 40G,and FIG. 40K): NK cells; lower right panel (in FIG. 40D, FIG. 40H, andFIG. 40L): CD14⁺ cells (monocytes/neutrophils).

DETAILED DESCRIPTION OF THE INVENTION Definitions

Terms are used herein as generally used in the art, unless otherwisedefined in the following.

As used herein, the term “conjugate” refers to a fusion polypeptidemolecule that includes one effector moiety and a further peptidemolecule, particularly an immunoglobulin molecule.

As used herein, the term “immunoconjugate” refers to a fusionpolypeptide molecule that includes one effector moiety, at least oneantigen binding moiety and an Fc domain, provided that not more than oneeffector moiety is present. In certain embodiments, the immunoconjugatecomprises one effector moiety, two antigen binding moieties, and an Fcdomain. Particular immunoconjugates according to the inventionessentially consist of one effector moiety, two antigen bindingmoieties, and an Fc domain, joined by one or more linker sequences. Theantigen binding moiety and the effector moiety can be joined to the Fcdomain by a variety of interactions and in a variety of configurationsas described herein. In a particular embodiment, the two antigen bindingmoieties and the Fc domain are joined to each other in a configurationso as to form a full immunoglobulin molecule. An immunoconjugate asreferred to herein, is a fusion protein, i.e. the components of theimmunoconjugate are linked to each other by peptide-bonds, eitherdirectly or through linker peptides.

As used herein, the term “antigen binding moiety” refers to apolypeptide molecule that specifically binds to an antigenicdeterminant. In one embodiment, an antigen binding moiety is able todirect the entity to which it is attached (e.g. an effector moiety or asecond antigen binding moiety) to a target site, for example to aspecific type of tumor cell or tumor stroma bearing the antigenicdeterminant. Antigen binding moieties include antibodies and fragmentsthereof as further defined herein. Particular antigen binding moietiesinclude an antigen binding domain of an antibody, comprising an antibodyheavy chain variable region and an antibody light chain variable region.In certain embodiments, the antigen binding moieties may compriseantibody constant regions as further defined herein and known in theart. Useful heavy chain constant regions include any of the fiveisotypes: α, δ, ε, γ, or μ. Useful light chain constant regions includeany of the two isotypes: κ and λ.

As used herein, the term “antigenic determinant” is synonymous with“antigen” and “epitope,” and refers to a site (e.g. a contiguous stretchof amino acids or a conformational configuration made up of differentregions of non-contiguous amino acids) on a polypeptide macromolecule towhich an antigen binding moiety binds, forming an antigen bindingmoiety-antigen complex.

Useful antigenic determinants can be found, for example, on the surfacesof tumor cells, on the surfaces of virus-infected cells, on the surfacesof other diseased cells, free in blood serum, and/or in theextracellular matrix (ECM). In a particular embodiment the antigenicdeterminant is a human antigen.

By “specifically binds” is meant that the binding is selective for theantigen and can be discriminated from unwanted or non-specificinteractions. The ability of an antigen-binding moiety to bind to aspecific antigenic determinant can be measured either through anenzyme-linked immunosorbent assay (ELISA) or other techniques familiarto one of skill in the art, e.g. surface plasmon resonance (SPR)technique (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res28, 217-229 (2002)). In one embodiment, the extent of binding of anantigen binding moiety to an unrelated protein is less than about 10% ofthe binding of the antigen binding moiety to the antigen as measured,e.g., by SPR. In certain embodiments, an antigen binding moiety thatbinds to the antigen, or an immunoconjugate comprising that antigenbinding moiety, has a dissociation constant (K_(D)) of ≦1 μM, ≦100 nM,≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g. 10⁻⁸M or less, e.g.from 10⁻⁸M to 10⁻¹³M, e.g., from 10⁻⁹M to 10⁻¹³ M).

“Affinity” refers to the strength of the sum total of non-covalentinteractions between a single binding site of a molecule (e.g., areceptor) and its binding partner (e.g., a ligand). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., receptor and a ligand). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (K_(D)), which is the ratio of dissociation and associationrate constants (k_(off) and k_(on), respectively). Thus, equivalentaffinities may comprise different rate constants, as long as the ratioof the rate constants remains the same. Affinity can be measured by wellestablished methods known in the art, including those described herein.A particular method for measuring affinity is Surface Plasmon Resonance(SPR).

“Reduced binding”, for example reduced binding to an Fc receptor or toCD25, refers to a decrease in affinity for the respective interaction,as measured for example by SPR. For clarity the term includes alsoreduction of the affinity to zero (or below the detection limit of theanalytic method), i.e. complete abolishment of the interaction.Conversely, “increased binding” refers to an increase in bindingaffinity for the respective interaction.

As used herein, the terms “first” and “second” with respect toantigen-binding moieties etc., are used for convenience ofdistinguishing when there is more than one of each type of moiety. Useof these terms is not intended to confer a specific order or orientationof the immunoconjugate unless explicitly so stated.

As used herein, the term “effector moiety” refers to a polypeptide,e.g., a protein or glycoprotein, that influences cellular activity, forexample, through signal transduction or other cellular pathways.Accordingly, the effector moiety of the invention can be associated withreceptor-mediated signaling that transmits a signal from outside thecell membrane to modulate a response in a cell bearing one or morereceptors for the effector moiety. In one embodiment, an effector moietycan elicit a cytotoxic response in cells bearing one or more receptorsfor the effector moiety. In another embodiment, an effector moiety canelicit a proliferative response in cells bearing one or more receptorsfor the effector moiety. In another embodiment, an effector moiety canelicit differentiation in cells bearing receptors for the effectormoiety. In another embodiment, an effector moiety can alter expression(i.e. upregulate or downregulate) of an endogenous cellular protein incells bearing receptors for the effector moiety. Non-limiting examplesof effector moieties include cytokines, growth factors, hormones,enzymes, substrates, and cofactors. The effector moiety can beassociated with an antigen-binding moiety or an Fc domain in a varietyof configurations to form an immunoconjugate.

As used herein, the term “cytokine” refers to a molecule that mediatesand/or regulates a biological or cellular function or process (e.g.immunity, inflammation, and hematopoiesis). The term “cytokine” as usedherein includes “lymphokines,” “chemokines,” “monokines,” and“interleukins”. Examples of useful cytokines include, but are notlimited to, GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-10, IL-12, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNF-α,and TNF-β. Particular cytokines are IL-2, IL-7, IL-10, IL-12, IL-15,IFN-α and IFN-γ. In particular embodiments the cytokine is a humancytokine. The term “cytokine” as used herein is meant to also includecytokine variants comprising one or more amino acid mutations in theamino acid sequences of the corresponding wild-type cytokine, such asfor example the IL-2 variants described in Sauvé et al., Proc Natl AcadSci USA 88, 4636-40 (1991); Hu et al., Blood 101, 4853-4861 (2003) andUS Pat. Publ. No. 2003/0124678; Shanafelt et al., Nature Biotechnol 18,1197-1202 (2000); Heaton et al., Cancer Res 53, 2597-602 (1993) and U.S.Pat. No. 5,229,109; US Pat. Publ. No. 2007/0036752; WO 2008/0034473; WO2009/061853; or PCT patent application no. PCT/EP2012/051991. Furthercytokine variants, for example variants of IL-15, are described herein.In certain embodiments cytokines have been mutated to eliminateglycosylation.

As used herein, the term “single-chain” refers to a molecule comprisingamino acid monomers linearly linked by peptide bonds. In one embodiment,the effector moiety is a single-chain effector moiety. Non-limitingexamples of single-chain effector moieties include cytokines, growthfactors, hormones, enzymes, substrates, and cofactors. When the effectormoiety is a cytokine and the cytokine of interest is normally found as amultimer in nature, each subunit of the multimeric cytokine issequentially encoded by the single-chain of the effector moiety.Accordingly, non-limiting examples of useful single-chain effectormoieties include GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-10, IL-12, IL-15, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β,TGF-β, TNF-α, and TNF-β.

As used herein, the term “control effector moiety” refers to anunconjugated effector moiety. For example, when comparing an IL-2immunoconjugate as described herein with a control effector moiety, thecontrol effector moiety is free, unconjugated IL-2. Likewise, e.g., whencomparing an IL-12 immunoconjugate with a control effector moiety, thecontrol effector moiety is free, unconjugated IL-12 (e.g. existing as aheterodimeric protein wherein the p40 and p35 subunits share onlydisulfide bond(s)).

As used herein, the term “effector moiety receptor” refers to apolypeptide molecule capable of binding specifically to an effectormoiety. For example, where IL-2 is the effector moiety, the effectormoiety receptor that binds to an IL-2 molecule (e.g. an immunoconjugatecomprising IL-2) is the IL-2 receptor. Similarly, e.g., where IL-12 isthe effector moiety of an immunoconjugate, the effector moiety receptoris the IL-12 receptor. Where an effector moiety specifically binds tomore than one receptor, all receptors that specifically bind to theeffector moiety are “effector moiety receptors” for that effectormoiety.

The term “immunoglobulin molecule” refers to a protein having thestructure of a naturally occurring antibody. For example,immunoglobulins of the IgG class are heterotetrameric glycoproteins ofabout 150,000 daltons, composed of two light chains and two heavy chainsthat are disulfide-bonded. From N- to C-terminus, each heavy chain has avariable region (VH), also called a variable heavy domain or a heavychain variable domain, followed by three constant domains (CH1, CH2, andCH3), also called a heavy chain constant region. Similarly, from N- toC-terminus, each light chain has a variable region (VL), also called avariable light domain or a light chain variable domain, followed by aconstant light (CL) domain, also called a light chain constant region.The heavy chain of an immunoglobulin may be assigned to one of fivetypes, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some ofwhich may be further divided into subtypes, e.g. γ₁ (IgG₁), γ₂ (IgG₂),γ₃ (IgG₃), γ₄ (IgG₄), α₁ (IgA₁) and α₂ (IgA₂). The light chain of animmunoglobulin may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain. Animmunoglobulin essentially consists of two Fab molecules and an Fcdomain, linked via the immunoglobulin hinge region.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, and antibody fragments so long asthey exhibit the desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂, diabodies, linear antibodies, single-chain antibody molecules(e.g. scFv), and single-domain antibodies. For a review of certainantibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For areview of scFv fragments, see e.g. Plückthun, in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93/16185; andU.S. Pat. Nos. 5,571,894 and 5,587,458. For discussion of Fab andF(ab′)₂ fragments comprising salvage receptor binding epitope residuesand having increased in vivo half-life, see U.S. Pat. No. 5,869,046.Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., ProcNatl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies arealso described in Hudson et al., Nat Med 9, 129-134 (2003).Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see e.g. U.S. Pat. No. 6,248,516 B1). Antibodyfragments can be made by various techniques, including but not limitedto proteolytic digestion of an intact antibody as well as production byrecombinant host cells (e.g. E. coli or phage), as described herein.

The term “antigen binding domain” refers to the part of an antibody thatcomprises the area which specifically binds to and is complementary topart or all of an antigen. An antigen binding domain may be provided by,for example, one or more antibody variable domains (also called antibodyvariable regions). Particularly, an antigen binding domain comprises anantibody light chain variable region (VL) and an antibody heavy chainvariable region (VH).

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindtet al., Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007). A single VH or VL domain may be sufficient to conferantigen-binding specificity.

The term “hypervariable region” or “HVR”, as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe complementarity determining regions (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.

With the exception of CDR1 in VH, CDRs generally comprise the amino acidresidues that form the hypervariable loops. Hypervariable regions (HVRs)are also referred to as “complementarity determining regions” (CDRs),and these terms are used herein interchangeably in reference to portionsof the variable region that form the antigen binding regions. Thisparticular region has been described by Kabat et al., U.S. Dept. ofHealth and Human Services, Sequences of Proteins of ImmunologicalInterest (1983) and by Chothia et al., J Mol Biol 196:901-917 (1987),where the definitions include overlapping or subsets of amino acidresidues when compared against each other. Nevertheless, application ofeither definition to refer to a CDR of an antibody or variants thereofis intended to be within the scope of the term as defined and usedherein. The appropriate amino acid residues which encompass the CDRs asdefined by each of the above cited references are set forth below inTable 1 as a comparison. The exact residue numbers which encompass aparticular CDR will vary depending on the sequence and size of the CDR.Those skilled in the art can routinely determine which residues comprisea particular CDR given the variable region amino acid sequence of theantibody.

TABLE 1 CDR Definitions¹ CDR Kabat Chothia AbM² V_(H) CDR1 31-35 26-3226-35 V_(H) CDR2 50-65 52-58 50-58 V_(H) CDR3 95-102 95-102 95-102 V_(L)CDR1 24-34 26-32 24-34 V_(L) CDR2 50-56 50-52 50-56 V_(L) CDR3 89-9791-96 89-97 ¹Numbering of all CDR definitions in Table 1 is according tothe numbering conventions set forth by Kabat et al. (see below). ²“AbM”with a lowercase “b” as used in Table 1 refers to the CDRs as defined byOxford Molecular's “AbM” antibody modeling software.

Kabat et al. also defined a numbering system for variable regionsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable region sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in an antibody variable region areaccording to the Kabat numbering system.

The polypeptide sequences of the sequence listing (i.e., SEQ ID NOs 23,25, 27, 29, 31, etc.) are not numbered according to the Kabat numberingsystem. However, it is well within the ordinary skill of one in the artto convert the numbering of the sequences of the Sequence Listing toKabat numbering.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The “class” of an antibody or immunoglobulin refers to the type ofconstant domain or constant region possessed by its heavy chain. Thereare five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constantdomains that correspond to the different classes of immunoglobulins arecalled α, δ, ε, γ, and μ, respectively.

The term “Fc domain” or “Fc region” herein is used to define aC-terminal region of an immunoglobulin heavy chain that contains atleast a portion of the constant region. The term includes nativesequence Fc regions and variant Fc regions. Although the boundaries ofthe Fc region of an IgG heavy chain might vary slightly, the human IgGheavy chain Fc region is usually defined to extend from Cys226, or fromPro230, to the carboxyl-terminus of the heavy chain. However, theC-terminal lysine (Lys447) of the Fc region may or may not be present.Unless otherwise specified herein, numbering of amino acid residues inthe Fc region or constant region is according to the EU numberingsystem, also called the EU index, as described in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md., 1991. A “subunit”of an Fc domain as used herein refers to one of the two polypeptidesforming the dimeric Fc domain, i.e. a polypeptide comprising C-terminalconstant regions of an immunoglobulin heavy chain, capable of stableself-association. For example, a subunit of an IgG Fc domain comprisesan IgG CH2 and an IgG CH3 constant domain.

A “modification promoting heterodimerization” is a manipulation of thepeptide backbone or the post-translational modifications of apolypeptide that reduces or prevents the association of the polypeptidewith an identical polypeptide to form a homodimer. A modificationpromoting heterodimerization as used herein particularly includesseparate modifications made to each of two polypeptides desired to forma dimer, wherein the modifications are complementary to each other so asto promote association of the two polypeptides. For example, amodification promoting heterodimerization may alter the structure orcharge of one or both of the polypeptides desired to form a dimer so asto make their association sterically or electrostatically favorable,respectively. Heterodimerization occurs between two non-identicalpolypeptides, such as two subunits of an Fc domain wherein furtherimmunoconjugate components fused to each of the subunits (e.g. antigenbinding moiety, effector moiety) are not the same. In theimmunoconjugates according to the present invention, the modificationpromoting heterodimerization is in the Fc domain. In some embodimentsthe modification promoting heterodimerziation comprises an amino acidmutation, specifically an amino acid substitution. In a particularembodiment, the modification promoting heterodimerization comprises aseparate amino acid mutation, specifically an amino acid substitution,in each of the two subunits of the Fc domain.

The term “effector functions” refers to those biological activitiesattributable to the Fc region of an antibody, which vary with theantibody isotype. Examples of antibody effector functions include: C1qbinding and complement dependent cytotoxicity (CDC), Fc receptorbinding, antibody-dependent cell-mediated cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP), cytokine secretion,immune complex-mediated antigen uptake by antigen presenting cells, downregulation of cell surface receptors (e.g. B cell receptor), and B cellactivation.

As used herein, the terms “engineer, engineered, engineering”, areconsidered to include any manipulation of the peptide backbone or thepost-translational modifications of a naturally occurring or recombinantpolypeptide or fragment thereof. Engineering includes modifications ofthe amino acid sequence, of the glycosylation pattern, or of the sidechain group of individual amino acids, as well as combinations of theseapproaches. “Engineering”, particularly with the prefix “glyco-”, aswell as the term “glycosylation engineering” includes metabolicengineering of the glycosylation machinery of a cell, including geneticmanipulations of the oligosaccharide synthesis pathways to achievealtered glycosylation of glycoproteins expressed in cells. Furthermore,glycosylation engineering includes the effects of mutations and cellenvironment on glycosylation. In one embodiment, the glycosylationengineering is an alteration in glycosyltransferase activity. In aparticular embodiment, the engineering results in alteredglucosaminyltransferase activity and/or fucosyltransferase activity.Glycosylation engineering can be used to obtain a “host cell havingincreased GnTIII activity”, a “host cell having increased ManIIactivity”, or a “host cell having decreased α(1,6) fucosyltransferaseactivity”.

The term “amino acid mutation” as used herein is meant to encompassamino acid substitutions, deletions, insertions, and modifications. Anycombination of substitution, deletion, insertion, and modification canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., reduced bindingto an Fc receptor, or reduced binding to CD25. Amino acid sequencedeletions and insertions include amino- and/or carboxy-terminaldeletions and insertions of amino acids. Particular amino acid mutationsare amino acid substitutions. For the purpose of altering e.g. thebinding characteristics of an Fc region or a cytokine such as IL-2,non-conservative amino acid substitutions, i.e. replacing one amino acidwith another amino acid having different structural and/or chemicalproperties, are particularly preferred. Amino acid substitutions includereplacement by non-naturally occurring amino acids or by naturallyoccurring amino acid derivatives of the twenty standard amino acids(e.g. 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine,5-hydroxylysine). Amino acid mutations can be generated using genetic orchemical methods well known in the art. Genetic methods may includesite-directed mutagenesis, PCR, gene synthesis and the like. It iscontemplated that methods of altering the side chain group of an aminoacid by methods other than genetic engineering, such as chemicalmodification, may also be useful. Various designations may be usedherein to indicate the same amino acid mutation. For example, asubstitution from proline at position 329 of the Fc domain to glycinecan be indicated as 329G, G329, G₃₂₉, P329G, or Pro329Gly.

As used herein, term “polypeptide” refers to a molecule composed ofmonomers (amino acids) linearly linked by amide bonds (also known aspeptide bonds). The term “polypeptide” refers to any chain of two ormore amino acids, and does not refer to a specific length of theproduct. Thus, peptides, dipeptides, tripeptides, oligopeptides,“protein,” “amino acid chain,” or any other term used to refer to achain of two or more amino acids, are included within the definition of“polypeptide,” and the term “polypeptide” may be used instead of, orinterchangeably with any of these terms. The term “polypeptide” is alsointended to refer to the products of post-expression modifications ofthe polypeptide, including without limitation glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, or modification bynon-naturally occurring amino acids. A polypeptide may be derived from anatural biological source or produced by recombinant technology, but isnot necessarily translated from a designated nucleic acid sequence. Itmay be generated in any manner, including by chemical synthesis. Apolypeptide of the invention may be of a size of about 3 or more, 5 ormore, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 ormore, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides may have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides which do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations, and arereferred to as unfolded.

By an “isolated” polypeptide or a variant, or derivative thereof isintended a polypeptide that is not in its natural milieu. No particularlevel of purification is required. For example, an isolated polypeptidecan be removed from its native or natural environment. Recombinantlyproduced polypeptides and proteins expressed in host cells areconsidered isolated for the purpose of the invention, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary. In situations where ALIGN-2 is employed for amino acidsequence comparisons, the % amino acid sequence identity of a givenamino acid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “polynucleotide” refers to an isolated nucleic acid molecule orconstruct, e.g. messenger RNA (mRNA), virally-derived RNA, or plasmidDNA (pDNA). A polynucleotide may comprise a conventional phosphodiesterbond or a non-conventional bond (e.g. an amide bond, such as found inpeptide nucleic acids (PNA). The term “nucleic acid molecule” refers toany one or more nucleic acid segments, e.g. DNA or RNA fragments,present in a polynucleotide.

By “isolated” nucleic acid molecule or polynucleotide is intended anucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotide encoding atherapeutic polypeptide contained in a vector is considered isolated forthe purposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. An isolated polynucleotide includes apolynucleotide molecule contained in cells that ordinarily contain thepolynucleotide molecule, but the polynucleotide molecule is presentextrachromosomally or at a chromosomal location that is different fromits natural chromosomal location. Isolated RNA molecules include in vivoor in vitro RNA transcripts of the present invention, as well aspositive and negative strand forms, and double-stranded forms. Isolatedpolynucleotides or nucleic acids according to the present inventionfurther include such molecules produced synthetically. In addition, apolynucleotide or a nucleic acid may be or may include a regulatoryelement such as a promoter, ribosome binding site, or a transcriptionterminator.

By a nucleic acid or polynucleotide having a nucleotide sequence atleast, for example, 95% “identical” to a reference nucleotide sequenceof the present invention, it is intended that the nucleotide sequence ofthe polynucleotide is identical to the reference sequence except thatthe polynucleotide sequence may include up to five point mutations pereach 100 nucleotides of the reference nucleotide sequence. In otherwords, to obtain a polynucleotide having a nucleotide sequence at least95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. These alterations of the reference sequence may occur at the5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence. As a practical matter,whether any particular polynucleotide sequence is at least 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of thepresent invention can be determined conventionally using known computerprograms, such as the ones discussed above for polypeptides (e.g.ALIGN-2).

The term “expression cassette” refers to a polynucleotide generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in atarget cell. The recombinant expression cassette can be incorporatedinto a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, ornucleic acid fragment. Typically, the recombinant expression cassetteportion of an expression vector includes, among other sequences, anucleic acid sequence to be transcribed and a promoter. In certainembodiments, the expression cassette of the invention comprisespolynucleotide sequences that encode immunoconjugates of the inventionor fragments thereof.

The term “vector” or “expression vector” is synonymous with “expressionconstruct” and refers to a DNA molecule that is used to introduce anddirect the expression of a specific gene to which it is operablyassociated in a target cell. The term includes the vector as aself-replicating nucleic acid structure as well as the vectorincorporated into the genome of a host cell into which it has beenintroduced. The expression vector of the present invention comprises anexpression cassette. Expression vectors allow transcription of largeamounts of stable mRNA. Once the expression vector is inside the targetcell, the ribonucleic acid molecule or protein that is encoded by thegene is produced by the cellular transcription and/or translationmachinery. In one embodiment, the expression vector of the inventioncomprises an expression cassette that comprises polynucleotide sequencesthat encode immunoconjugates of the invention or fragments thereof.

The term “artificial” refers to a synthetic, or non-host cell derivedcomposition, e.g. a chemically-synthesized oligonucleotide.

The terms “host cell”, “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages.

Progeny may not be completely identical in nucleic acid content to aparent cell, but may contain mutations. Mutant progeny that have thesame function or biological activity as screened or selected for in theoriginally transformed cell are included herein. A host cell is any typeof cellular system that can be used to generate the immunoconjugatesused for the present invention. In one embodiment, the host cell isengineered to allow the production of an immunoconjugate with modifiedoligosaccharides in its Fc region. In certain embodiments, the hostcells have been manipulated to express increased levels of one or morepolypeptides having β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)activity. In certain embodiments the host cells have been furthermanipulated to express increased levels of one or more polypeptideshaving α-mannosidase II (ManII) activity. Host cells include culturedcells, e.g. mammalian cultured cells, such as CHO cells, BHK cells, NS0cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PERcells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, andplant cells, to name only a few, but also cells comprised within atransgenic animal, transgenic plant or cultured plant or animal tissue.

As used herein, the term “polypeptide having GnTIII activity” refers topolypeptides that are able to catalyze the addition of aN-acetylglucosamine (GlcNAc) residue in β-1,4 linkage to the β-linkedmannoside of the trimannosyl core of N-linked oligosaccharides. Thisincludes fusion polypeptides exhibiting enzymatic activity similar to,but not necessarily identical to, an activity ofβ(1,4)-N-acetylglucosaminyltransferase III, also known asβ-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyl-transferase (EC2.4.1.144), according to the Nomenclature Committee of the InternationalUnion of Biochemistry and Molecular Biology (NC-IUBMB), as measured in aparticular biological assay, with or without dose dependency. In thecase where dose dependency does exist, it need not be identical to thatof GnTIII, but rather substantially similar to the dose-dependency in agiven activity as compared to the GnTIII (i.e. the candidate polypeptidewill exhibit greater activity or not more than about 25-fold less and,preferably, not more than about ten-fold less activity, and mostpreferably, not more than about three-fold less activity relative to theGnTIII). In certain embodiments the polypeptide having GnTIII activityis a fusion polypeptide comprising the catalytic domain of GnTIII andthe Golgi localization domain of a heterologous Golgi residentpolypeptide. Particularly, the Golgi localization domain is thelocalization domain of mannosidase II or GnTI, most particularly thelocalization domain of mannosidase II. Alternatively, the Golgilocalization domain is selected from the group consisting of: thelocalization domain of mannosidase I, the localization domain of GnTII,and the localization domain of α1,6 core fucosyltransferase. Methods forgenerating such fusion polypeptides and using them to produce antibodieswith increased effector functions are disclosed in WO 2004/065540, U.S.Provisional Pat. Appl. No. 60/495,142 and U.S. Pat. Appl. Publ. No.2004/0241817, the entire contents of which are expressly incorporatedherein by reference.

As used herein, the term “Golgi localization domain” refers to the aminoacid sequence of a Golgi resident polypeptide which is responsible foranchoring the polypeptide to a location within the Golgi complex.Generally, localization domains comprise amino terminal “tails” of anenzyme.

As used herein, the term “polypeptide having ManII activity” refers topolypeptides that are able to catalyze the hydrolysis of the terminal1,3- and 1,6-linked α-D-mannose residues in the branchedGlcNAcMan₅GlcNAc₂ mannose intermediate of N-linked oligosaccharides.This includes polypeptides exhibiting enzymatic activity similar to, butnot necessarily identical to, an activity of Golgi α-mannosidase II,also known as mannosyl oligosaccharide 1,3-1,6-α-mannosidase II (EC3.2.1.114), according to the Nomenclature Committee of the InternationalUnion of Biochemistry and Molecular Biology (NC-IUBMB).

An “activating Fc receptor” is an Fc receptor that following engagementby an Fc region of an antibody (or immunoconjugate) elicits signalingevents that stimulate the receptor-bearing cell to perform effectorfunctions. Activating Fc receptors include FcγRIIIa (CD16a), FcγRI(CD64), FcγRIIa (CD32), and FcαRI (CD89).

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immunemechanism leading to the lysis of antibody-coated target cells by immuneeffector cells. The target cells are cells to which antibodies,immunoconjugates or fragments thereof comprising an Fc regionspecifically bind, generally via the protein part that is N-terminal tothe Fc region. As used herein, the term “increased ADCC” is defined aseither an increase in the number of target cells that are lysed in agiven time, at a given concentration of immunoconjugate in the mediumsurrounding the target cells, by the mechanism of ADCC defined above,and/or a reduction in the concentration of immunoconjugate, in themedium surrounding the target cells, required to achieve the lysis of agiven number of target cells in a given time, by the mechanism of ADCC.The increase in ADCC is relative to the ADCC mediated by the sameimmunoconjugate produced by the same type of host cells, using the samestandard production, purification, formulation and storage methods(which are known to those skilled in the art), but that has not beenengineered. For example the increase in ADCC mediated by animmunoconjugate produced by host cells engineered to have an alteredpattern of glycosylation (e.g. to express the glycosyltransferase,GnTIII, or other glycosyltransferases) by the methods described herein,is relative to the ADCC mediated by the same immunoconjugate produced bythe same type of non-engineered host cells.

By “immunoconjugate having increased antibody dependent cell-mediatedcytotoxicity (ADCC)” is meant an immunoconjugate having increased ADCCas determined by any suitable method known to those of ordinary skill inthe art. One accepted in vitro ADCC assay is as follows:

-   -   1) the assay uses target cells that are known to express the        target antigen recognized by the antigen binding moiety of the        immunoconjugate;    -   2) the assay uses human peripheral blood mononuclear cells        (PBMCs), isolated from blood of a randomly chosen healthy donor,        as effector cells;    -   3) the assay is carried out according to following protocol:    -   i) the PBMCs are isolated using standard density centrifugation        procedures and are suspended at 5×10⁶ cells/ml in RPMI cell        culture medium;    -   ii) the target cells are grown by standard tissue culture        methods, harvested from the exponential growth phase with a        viability higher than 90%, washed in RPMI cell culture medium,        labeled with 100 micro-Curies of ⁵¹Cr, washed twice with cell        culture medium, and resuspended in cell culture medium at a        density of 10⁵ cells/ml;    -   iii) 100 microliters of the final target cell suspension above        are transferred to each well of a 96-well microtiter plate;    -   iv) the immunoconjugate is serially-diluted from 4000 ng/ml to        0.04 ng/ml in cell culture medium and 50 microliters of the        resulting immunoconjugate solutions are added to the target        cells in the 96-well microtiter plate, testing in triplicate        various immunoconjugate concentrations covering the whole        concentration range above;    -   v) for the maximum release (MR) controls, 3 additional wells in        the plate containing the labeled target cells, receive 50        microliters of a 2% (V/V) aqueous solution of non-ionic        detergent (Nonidet, Sigma, St. Louis), instead of the        immunoconjugate solution (point iv above);    -   vi) for the spontaneous release (SR) controls, 3 additional        wells in the plate containing the labeled target cells, receive        50 microliters of RPMI cell culture medium instead of the        immunoconjugate solution (point iv above);    -   vii) the 96-well microtiter plate is then centrifuged at 50×g        for 1 minute and incubated for 1 hour at 4° C.;    -   viii) 50 microliters of the PBMC suspension (point i above) are        added to each well to yield an effector:target cell ratio of        25:1 and the plates are placed in an incubator under 5% CO₂        atmosphere at 37° C. for 4 hours;    -   ix) the cell-free supernatant from each well is harvested and        the experimentally released radioactivity (ER) is quantified        using a gamma counter;    -   x) the percentage of specific lysis is calculated for each        immunoconjugate concentration according to the formula        (ER−MR)/(MR−SR)×100, where ER is the average radioactivity        quantified (see point ix above) for that immunoconjugate        concentration, MR is the average radioactivity quantified (see        point ix above) for the MR controls (see point v above), and SR        is the average radioactivity quantified (see point ix above) for        the SR controls (see point vi above);    -   4) “increased ADCC” is defined as either an increase in the        maximum percentage of specific lysis observed within the        immunoconjugate concentration range tested above, and/or a        reduction in the concentration of immunoconjugate required to        achieve one half of the maximum percentage of specific lysis        observed within the immunoconjugate concentration range tested        above. The increase in ADCC is relative to the ADCC, measured        with the above assay, mediated by the same immunoconjugate,        produced by the same type of host cells, using the same standard        production, purification, formulation and storage methods, which        are known to those skilled in the art, but that has not been        engineered.

An “effective amount” of an agent refers to the amount that is necessaryto result in a physiological change in the cell or tissue to which it isadministered.

A “therapeutically effective amount” of an agent, e.g. a pharmaceuticalcomposition, refers to an amount effective, at dosages and for periodsof time necessary, to achieve the desired therapeutic or prophylacticresult. A therapeutically effective amount of an agent for exampleeliminates, decreases, delays, minimizes or prevents adverse effects ofa disease.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g. cows, sheep, cats, dogs, andhorses), primates (e.g. humans and non-human primates such as monkeys),rabbits, and rodents (e.g. mice and rats). Particularly, the individualor subject is a human.

The term “pharmaceutical composition” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical composition, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of a disease in the individual being treated,and can be performed either for prophylaxis or during the course ofclinical pathology. Desirable effects of treatment include, but are notlimited to, preventing occurrence or recurrence of disease, alleviationof symptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments,immunoconjugates of the invention are used to delay development of adisease or to slow the progression of a disease.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

DETAILED DESCRIPTION OF THE EMBODIMENTS

In a first aspect the invention provides an immunoconjugate comprising afirst antigen binding moiety, an Fc domain consisting of two subunits,and an effector moiety, wherein not more than one effector moiety ispresent. The absence of further effector moieties may reduce targetingof the immunoconjugate to sites where the respective effector moietyreceptor is presented, thereby improving targeting to and accumulationat sites where the actual target antigen of the immunoconjugate, whichis recognized by the antigen binding moiety, is presented. Furthermore,the absence of an avidity effect for the respective effector moietyreceptor can reduce activation of effector moiety receptor-positivecells in peripheral blood upon intravenous administration of theimmunoconjugate. Furthermore, the serum half-life of immunoconjugatescomprising only a single effector moiety appears to be longer ascompared to immunoconjugates comprising two or more effector moieties.

Immunoconjugate Formats

The components of the immunoconjugate can be fused to each other in avariety of configurations. Exemplary configurations are depicted inFIGS. 2A-C. In one embodiment the effector moiety is fused to the amino-or carboxy-terminal amino acid of one of the two subunits of the Fcdomain. In one embodiment the effector moiety is fused to thecarboxy-terminal amino acid of one of the two subunits of the Fc domain.The effector moiety may be fused to the Fc domain directly or through alinker peptide, comprising one or more amino acids, typically about 2-20amino acids. Linker peptides are known in the art or are describedherein. Suitable, non-immunogenic linker peptides include, for example,(G₄S)_(n), (SG₄)_(n) or G₄(SG₄)_(n) linker peptides. “n” is generally anumber between 1 and 10, typically between 2 and 4. Alternatively, wherethe effector moiety is linked to the N-terminus of an Fc domain subunit,it may be linked via an immunoglobulin hinge region or a portionthereof, with or without an additional linker peptide.

Similarly, the first antigen binding moiety can be fused to the amino-or carboxy-terminal amino acid of one of the two subunits of the Fcdomain. In one embodiment the first antigen binding moiety is fused tothe amino-terminal amino acid of one of the two subunits of the Fcdomain.

The first antigen binding moiety may be fused to the Fc domain directlyor through a linker peptide. In a particular embodiment the firstantigen binding moiety is fused to the Fc domain through animmunoglobulin hinge region. In a specific embodiment, theimmunoglobulin hinge region is a human IgG₁ hinge region.

In one embodiment the first antigen binding moiety comprises an antigenbinding domain of an antibody, comprising an antibody heavy chainvariable region and an antibody light chain variable region. In aparticular embodiment the first antigen binding moiety is a Fabmolecule. In one embodiment the Fab molecule is fused at its heavy orlight chain carboxy-terminus to the amino-terminal amino acid of one ofthe two subunits of the Fc domain. In a particular embodiment the Fabmolecule is fused at its heavy chain carboxy-terminus to theamino-terminal amino acid of one of the two subunits of the Fc domain.In a more particular embodiment the Fab molecule is fused to the Fcdomain through an immunoglobulin hinge region. In a specific embodiment,the immunoglobulin hinge region is a human IgG₁ hinge region.

In one embodiment the immunoconjugate essentially consists of an antigenbinding moiety, an Fc domain consisting of two subunits, an effectormoiety, and optionally one or more linker peptides, wherein said antigenbinding domain is a Fab molecule and is fused at its heavy chaincarboxy-terminus to the amino-terminal amino acid of one of the twosubunits of the Fc domain, and wherein said effector moiety is fusedeither (i) to the amino-terminal amino acid of the other one of the twosubunits of the Fc domain, or (ii) to the carboxy-terminal amino acid ofone of the two subunits of the Fc domain. In the latter case, theeffector moiety and the first antigen binding moiety may both be fusedto the same subunit of the Fc domain, or may each be fused to adifferent one of the two subunits of the Fc domain.

An immunoconjugate format with a single antigen binding moiety (forexample as shown in FIGS. 2B and 2C) is useful, particularly in caseswhere internalization of the target antigen is to be expected followingbinding of a high affinity antigen binding moiety. In such cases, thepresence of more than one antigen binding moiety per immunoconjugate mayenhance internalization, thereby reducing availability of the targetantigen.

In many other cases, however, it will be advantageous to have animmunoconjugate comprising two or more antigen binding moieties and asingle effector moiety to optimize targeting to the target antigenversus the effector moiety receptor, and the pharmaceutical window ofthe immunoconjugate.

Thus, in a particular embodiment the immunoconjugate of the inventioncomprises a first and a second antigen binding moiety. In one embodimenteach of said first and second antigen binding moieties is fused to theamino-terminal amino acid of one of the two subunits of the Fc domain.

The first and second antigen binding moieties may be fused to the Fcdomain directly or through a linker peptide. In a particular embodimenteach of said first and second antigen binding moieties is fused to asubunit of the Fc domain through an immunoglobulin hinge region. In aspecific embodiment, the immunoglobulin hinge region is a human IgG₁hinge region.

In one embodiment each of said first and second antigen binding moietiescomprises an antigen binding domain of an antibody, comprising anantibody heavy chain variable region and an antibody light chainvariable region. In a particular embodiment each of said first andsecond antigen binding moieties is a Fab molecule. In one embodimenteach of said Fab molecules is fused at its heavy or light chaincarboxy-terminus to the amino-terminal amino acid of one of the twosubunits of the Fc domain. In a particular embodiment each of said Fabmolecules is fused at its heavy chain carboxy-terminus to theamino-terminal amino acid of one of the two subunits of the Fc domain.In a more particular embodiment each of said Fab molecules is fused to asubunit of the Fc domain through an immunoglobulin hinge region. In aspecific embodiment, the immunoglobulin hinge region is a human IgG₁hinge region.

In one embodiment the first and the second antigen binding moiety andthe Fc domain are part of an immunoglobulin molecule. In a particularembodiment the immunoglobulin molecule is an IgG class immunoglobulin.In an even more particular embodiment the immunoglobulin is an IgG₁subclass immunoglobulin. In another particular embodiment theimmunoglobulin is a human immunoglobulin. In other embodiments theimmunoglobulin is a chimeric immunoglobulin or a humanizedimmunoglobulin. In one embodiment the effector moiety is fused to thecarboxy-terminal amino acid of one of the immunoglobulin heavy chains.The effector moiety may be fused to the immunoglobulin heavy chaindirectly or through a linker peptide. In a particular embodiment theimmunoconjugate essentially consists of an immunoglobulin molecule, aneffector moiety fused to the carboxy-terminal amino acid of one of theimmunoglobulin heavy chains, and optionally one or more linker peptides.

In one embodiment the immunoconjugate comprises a polypeptide wherein aFab heavy chain shares a carboxy-terminal peptide bond with an Fc domainsubunit and a polypeptide wherein an Fc domain subunit shares acarboxy-terminal peptide bond with an effector moiety polypeptide.

In another embodiment, the immunoconjugate comprises a polypeptidewherein a first Fab heavy chain shares a carboxy-terminal peptide bondwith an Fc domain subunit, and a polypeptide wherein a second Fab heavychain shares a carboxy-terminal peptide bond with an Fc domain subunit,which in turn shares a carboxy-terminal peptide bond with an effectormoiety polypeptide. In a further embodiment the immunoconjugatecomprises a polypeptide wherein a Fab heavy chain shares acarboxy-terminal peptide bond with an Fc domain subunit and apolypeptide wherein an effector moiety polypeptide shares acarboxy-terminal peptide bond with an Fc domain subunit. In someembodiments the immunoconjugate further comprises a Fab light chainpolypeptide. In certain embodiments the polypeptides are covalentlylinked, e.g., by a disulfide bond.

According to any of the above embodiments, components of theimmunoconjugate (e.g. effector moiety, antigen binding moiety, Fcdomain) may be linked directly or through various linkers, particularlypeptide linkers comprising one or more amino acids, typically about 2-20amino acids, that are described herein or are known in the art.Suitable, non-immunogenic linker peptides include, for example,(G4S)_(n), (SG₄)_(n) or G₄(SG₄)_(n) linker peptides, wherein n isgenerally a number between 1 and 10, typically between 2 and 4.

Fc Domain

The Fc domain of the immunoconjugate consists of a pair of polypeptidechains comprising heavy chain domains of an immunoglobulin molecule. Forexample, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer,each subunit of which comprises the CH2 and CH3 IgG heavy chain constantdomains. The two subunits of the Fc domain are capable of stableassociation with each other. In one embodiment the immunoconjugate ofthe invention comprises not more than one Fc domain.

In one embodiment according the invention the Fc domain of theimmunoconjugate is an IgG Fc domain. In a particular embodiment the Fcdomain is an IgG₁ Fc domain. In another embodiment, the Fc domain is anIgG₄ Fc domain. In a further particular embodiment the Fc domain ishuman. An exemplary sequence of a human IgG₁ Fc region is given in SEQID NO: 1.

The Fc domain confers to the immunoconjugate a greatly prolongedserum-half life as compared to immunoconjugate formats lacking an Fcdomain. Particularly when the immunoconjugate comprises an effectormoiety of rather weak activity (but e.g. reduced toxicity), a longhalf-life might be essential to achieve optimal efficacy in vivo.Moreover, the Fc domain can mediate effector functions, as will befurther discussed below.

Fc Domain Modifications Promoting Heterodimerization

Immunoconjugates according to the invention comprise only one singleeffector moiety, fused to one of the two subunits of the Fc domain, thusthey comprise two non-identical polypeptide chains. Recombinantco-expression of these polypeptides and subsequent dimerization leads toseveral possible combinations of the two polypeptides, out of which onlyheterodimers of the two non-identical polypeptides are useful accordingto the invention. To improve the yield and purity of immunoconjugates inrecombinant production, it can thus be advantageous to introduce in theFc domain of the immunoconjugate a modification which hinders theformation of homodimers of two identical polypeptides (i.e. twopolypeptides comprising an effector moiety, or two polypeptides lackingan effector moiety) and/or promotes the formation of heterodimers of apolypeptide comprising an effector moiety and a polypeptide lacking aneffector moiety.

Accordingly, in certain embodiments according to the invention the Fcdomain of the immunoconjugate comprises a modification promotingheterodimerization of two non-identical polypeptide chains. The site ofmost extensive protein-protein interaction between the two polypeptidechains of a human IgG Fc domain is in the CH3 domain of the Fc domain.Thus, in one embodiment said modification is in the CH3 domain of the Fcdomain.

In a specific embodiment said modification is a knob-into-holemodification, comprising a knob modification in one of the two subunitsof the Fc domain and a hole modification in the other one of the twosubunits of the Fc domain.

The knob-into-hole technology is described e.g. in U.S. Pat. No.5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621(1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, themethod involves introducing a protuberance (“knob”) at the interface ofa first polypeptide and a corresponding cavity (“hole”) in the interfaceof a second polypeptide, such that the protuberance can be positioned inthe cavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine). The protuberance and cavitycan be made by altering the nucleic acid encoding the polypeptides, e.g.by site-specific mutagenesis, or by peptide synthesis. In a specificembodiment a knob modification comprises the amino acid substitutionT366W in one of the two subunits of the Fc domain, and the holemodification comprises the amino acid substitutions T366S, L368A andY407V in the other one of the two subunits of the Fc domain. In afurther specific embodiment, the subunit of the Fc domain comprising theknob modification additionally comprises the amino acid substitutionS354C, and the subunit of the Fc domain comprising the hole modificationadditionally comprises the amino acid substitution Y349C. Introductionof these two cysteine residues results in formation of a disulfidebridge between the two subunits of the Fc region, further stabilizingthe dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

In an alternative embodiment a modification promoting heterodimerizationof two non-identical polypeptide chains comprises a modificationmediating electrostatic steering effects, e.g. as described in PCTpublication WO 2009/089004. Generally, this method involves replacementof one or more amino acid residues at the interface of the twopolypeptide chains by charged amino acid residues so that homodimerformation becomes electrostatically unfavorable but heterodimerizationelectrostatically favorable.

In a particular embodiment the effector moiety is fused to the amino- orcarboxy-terminal amino acid of the subunit of the Fc domain comprisingthe knob modification. Without wishing to be bound by theory, fusion ofthe effector moiety to the knob-containing subunit of the Fc domain willfurther minimize the generation of homodimeric immunoconjugatescomprising two effector moieties (steric clash of two knob-containingpolypeptides).

Fc Domain Modifications Altering Fc Receptor Binding

In certain embodiments of the invention the Fc domain of theimmunoconjugate is engineered to have altered binding affinity to an Fcreceptor, specifically altered binding affinity to an Fcγ receptor, ascompared to a non-engineered Fc domain.

Binding to Fc receptors can be easily determined e.g. by ELISA, or bySurface Plasmon Resonance (SPR) using standard instrumentation such as aBIAcore instrument (GE Healthcare), and Fc receptors such as may beobtained by recombinant expression. A suitable such binding assay isdescribed herein. Alternatively, binding affinity of Fc domains orimmunoconjugates comprising an Fc domain for Fc receptors may beevaluated using cell lines known to express particular Fc receptors,such as NK cells expressing FcγIIIa receptor.

In some embodiments the Fc domain of the immunoconjugate is engineeredto have altered effector functions, particularly altered ADCC, ascompared to a non-engineered Fc domain.

Effector function of an Fc domain, or an immunoconjugate comprising anFc domain, can be measured by methods known in the art. A suitable assayfor measuring ADCC is described herein. Other examples of in vitroassays to assess ADCC activity of a molecule of interest are describedin U.S. Pat. No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83,7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82,1499-1502 (1985); U.S. Pat. No. 5,821,337; Bruggemann et al., J Exp Med166, 1351-1361 (1987). Alternatively, non-radioactive assays methods maybe employed (see, for example, ACTI™ non-radioactive cytotoxicity assayfor flow cytometry (CellTechnology, Inc. Mountain View, Calif.); andCytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison,Wis.)). Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g. in a animal model such as that disclosed inClynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

In some embodiments binding of the Fc domain to a complement component,specifically to C1q, is altered. Accordingly, in some embodimentswherein the Fc domain is engineered to have altered effector function,said altered effector function includes altered CDC. C1q binding assaysmay be carried out to determine whether the immunoconjugate is able tobind C1q and hence has CDC activity. See e.g., C1q and C3c binding ELISAin WO 2006/029879 and WO 2005/100402. To assess complement activation, aCDC assay may be performed (see, for example, Gazzano-Santoro et al., JImmunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052(2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

a) Decreased Fc Receptor Binding and/or Effector Function

The Fc domain confers to the immunoconjugate favorable pharmacokineticproperties, including a long serum half-life which contributes to goodaccumulation in the target tissue and a favorable tissue-blooddistribution ratio. At the same time it may, however, lead toundesirable targeting of the immunoconjugate to cells expressing Fcreceptors rather than to the preferred antigen-bearing cells. Moreover,the co-activation of Fc receptor signaling pathways may lead to cytokinerelease which, in combination with the effector moiety and the longhalf-life of the immunoconjugate, results in excessive activation ofcytokine receptors and severe side effects upon systemic administration.In line with this, conventional IgG-IL-2 immunoconjugates have beendescribed to be associated with infusion reactions (see e.g. King etal., J Clin Oncol 22, 4463-4473 (2004)).

Accordingly, in particular embodiments according to the invention the Fcdomain of the immunoconjugate is engineered to have reduced bindingaffinity to an Fc receptor. In one such embodiment the Fc domaincomprises one or more amino acid mutation that reduces the bindingaffinity of the Fc domain to an Fc receptor. Typically, the same one ormore amino acid mutation is present in each of the two subunits of theFc domain. In one embodiment said amino acid mutation reduces thebinding affinity of the Fc domain to the Fc receptor by at least 2-fold,at least 5-fold, or at least 10-fold. In embodiments where there is morethan one amino acid mutation that reduces the binding affinity of the Fcdomain to the Fc receptor, the combination of these amino acid mutationsmay reduce the binding affinity of the Fc domain to the Fc receptor byat least 10-fold, at least 20-fold, or even at least 50-fold. In oneembodiment the immunoconjugate comprising an engineered Fc domainexhibits less than 20%, particularly less than 10%, more particularlyless than 5% of the binding affinity to an Fc receptor as compared to animmunoconjugate comprising a non-engineered Fc domain. In one embodimentthe Fc receptor is an activating Fc receptor. In a specific embodimentthe Fc receptor is an Fcγ receptor, more specifically an FcγRIIIa, FcγRIor FcγRIIa receptor. Preferably, binding to each of these receptors isreduced. In some embodiments binding affinity to a complement component,specifically binding affinity to C1q, is also reduced. In one embodimentbinding affinity to neonatal Fc receptor (FcRn) is not reduced.Substantially similar binding to FcRn, i.e. preservation of the bindingaffinity of the Fc domain to said receptor, is achieved when the Fcdomain (or the immunoconjugate comprising said Fc domain) exhibitsgreater than about 70% of the binding affinity of a non-engineered formof the Fc domain (or the immunoconjugate comprising said non-engineeredform of the Fc domain) to FcRn. Fc domains, or immunoconjugates of theinvention comprising said Fc domains, may exhibit greater than about 80%and even greater than about 90% of such affinity. In one embodiment theamino acid mutation is an amino acid substitution. In one embodiment theFc domain comprises an amino acid substitution at position P329. In amore specific embodiment the amino acid substitution is P329A or P329G,particularly P329G. In one embodiment the Fc domain comprises a furtheramino acid substitution at a position selected from 5228, E233, L234,L235, N297 and P331. In a more specific embodiment the further aminoacid substitution is S228P, E233P, L234A, L235A, L235E, N297A, N297D orP331 S. In a particular embodiment the Fc domain comprises amino acidsubstitutions at positions P329, L234 and L235. In a more particularembodiment the Fc domain comprises the amino acid mutations L234A, L235Aand P329G (LALA P329G). This combination of amino acid substitutionsalmost completely abolishes Fcγ receptor binding of a human IgG Fcdomain, as described in European patent application no. EP 11160251.2,incorporated herein by reference in its entirety. EP 11160251.2 alsodescribes methods of preparing such mutant Fc domains and methods fordetermining its properties such as Fc receptor binding or effectorfunctions.

Mutant Fc domains can be prepared by amino acid deletion, substitution,insertion or modification using genetic or chemical methods well knownin the art. Genetic methods may include site-specific mutagenesis of theencoding DNA sequence, PCR, gene synthesis, and the like. The correctnucleotide changes can be verified for example by sequencing.

In one embodiment the Fc domain is engineered to have decreased effectorfunction, compared to a non-engineered Fc domain. The decreased effectorfunction can include, but is not limited to, one or more of thefollowing: decreased complement dependent cytotoxicity (CDC), decreasedantibody-dependent cell-mediated cytotoxicity (ADCC), decreasedantibody-dependent cellular phagocytosis (ADCP), decreased cytokinesecretion, decreased immune complex-mediated antigen uptake byantigen-presenting cells, decreased binding to NK cells, decreasedbinding to macrophages, decreased binding to monocytes, decreasedbinding to polymorphonuclear cells, decreased direct signaling inducingapoptosis, decreased crosslinking of target-bound antibodies, decreaseddendritic cell maturation, or decreased T cell priming.

In one embodiment the decreased effector function is one or moreselected from the group of decreased CDC, decreased ADCC, decreasedADCP, and decreased cytokine secretion. In a particular embodiment thedecreased effector function is decreased ADCC. In one embodiment thedecreased ADCC is less than 20% of the ADCC induced by a non-engineeredFc domain (or an immunoconjugate comprising a non-engineered Fc domain).

In addition to the Fc domains described hereinabove and in Europeanpatent application no. EP 11160251.2, Fc domains with reduced Fcreceptor binding and/or effector function also include those withsubstitution of one or more of Fc domain residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

IgG₄ antibodies exhibit reduced binding affinity to Fc receptors andreduced effector functions as compared to IgG₁ antibodies. Hence, insome embodiments the Fc domain of the T cell activating bispecificantigen binding molecules of the invention is an IgG₄ Fc domain,particularly a human IgG₄ Fc domain. In one embodiment the IgG₄ Fcdomain comprises amino acid substitutions at position S228, specificallythe amino acid substitution S228P. To further reduce its bindingaffinity to an Fc receptor and/or its effector function, in oneembodiment the IgG₄ Fc domain comprises an amino acid substitution atposition L235, specifically the amino acid substitution L235E. Inanother embodiment, the IgG₄ Fc domain comprises an amino acidsubstitution at position P329, specifically the amino acid substitutionP329G. In a particular embodiment, the IgG₄ Fc domain comprises aminoacid substitutions at positions S228, L235 and P329, specifically aminoacid substitutions S228P, L235E and P329G. Such IgG₄ Fc domain mutantsand their Fcγ receptor binding properties are described in Europeanpatent application no. EP 11160251.2, incorporated herein by referencein its entirety.

b) Increased Fc Receptor Binding and/or Effector Function

Conversely, there may be situations where it is desirable to maintain oreven enhance Fc receptor binding and/or effector functions ofimmunoconjugates, for example when the immunoconjugate is targeted to ahighly specific tumor antigen. Hence, in certain embodiments the Fcdomain of the immunoconjugates of the invention is engineered to haveincreased binding affinity to an Fc receptor. Increased binding affinitymay be an increase in the binding affinity of the Fc domain to the Fcreceptor by at least 2-fold, at least 5-fold, or at least 10-fold. Inone embodiment the Fc receptor is an activating Fc receptor. In aspecific embodiment the Fc receptor is an Fcγ receptor. In oneembodiment the Fc receptor is selected from the group of FcγRIIIa, FcγRIand FcγRIIa. In a particular embodiment the Fc receptor is FcγRIIIa.

In one such embodiment the Fc domain is engineered to have an alteredoligosaccharide structure compared to a non-engineered Fc domain. In aparticular such embodiment the Fc domain comprises an increasedproportion of non-fucosylated oligosaccharides, compared to anon-engineered Fc domain. In a more specific embodiment, at least about20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%,about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about85%, about 90%, about 95%, or about 100%, particularly at least about50%, more particularly at least about 70%, of the N-linkedoligosaccharides in the Fc domain of the immunoconjugate arenon-fucosylated. The non-fucosylated oligosaccharides may be of thehybrid or complex type. In another specific embodiment the Fc domaincomprises an increased proportion of bisected oligosaccharides, comparedto a non-engineered Fc domain. In a more specific embodiment, at leastabout 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%,particularly at least about 50%, more particularly at least about 70%,of the N-linked oligosaccharides in the Fc domain of the immunoconjugateare bisected. The bisected oligosaccharides may be of the hybrid orcomplex type. In yet another specific embodiment the Fc domain comprisesan increased proportion of bisected, non-fucosylated oligosaccharides,compared to a non-engineered Fc domain. In a more specific embodiment,at least about 10%, about 15%, about 20%, about 25%, about 30%, about35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, orabout 100%, particularly at least about 15%, more particularly at leastabout 25%, at least about 35% or at least about 50%, of the N-linkedoligosaccharides in the Fc domain of the immunoconjugate are bisected,non-fucosylated. The bisected, non-fucosylated oligosaccharides may beof the hybrid or complex type.

The oligosaccharide structures in the immunoconjugate Fc domain can beanalysed by methods well known in the art, e.g. by MALDI TOF massspectrometry as described in Umana et al., Nat Biotechnol 17, 176-180(1999) or Ferrara et al., Biotechn Bioeng 93, 851-861 (2006). Thepercentage of non-fucosylated oligosaccharides is the amount ofoligosaccharides lacking fucose residues, relative to alloligosaccharides attached to Asn 297 (e.g. complex, hybrid and highmannose structures) and identified in an N-glycosidase F treated sampleby MALDI TOF MS. Asn 297 refers to the asparagine residue located atabout position 297 in the Fc domain (EU numbering of Fc regionresidues); however, Asn297 may also be located about ±3 amino acidsupstream or downstream of position 297, i.e., between positions 294 and300, due to minor sequence variations in immunoglobulins. The percentageof bisected, or bisected non-fucosylated, oligosaccharides is determinedanalogously.

Modification of the glycosylation in the Fc domain of theimmunoconjugate may result from production of the immunoconjugate in ahost cell that has been manipulated to express altered levels of one ormore polypeptides having glycosyltransferase activity.

In one embodiment the Fc domain of the immunoconjugate is engineered tohave an altered oligosaccharide structure, as compared to anon-engineered Fc domain, by producing the immunoconjugate in a hostcell having altered activity of one or more glycosyltransferase.Glycosyltransferases include for exampleβ(1,4)-N-acetylglucosaminyltransferase III (GnTIII),β(1,4)-galactosyltransferase (GalT),β(1,2)-N-acetylglucosaminyltransferase I (GnTI),β(1,2)-N-acetylglucosaminyltransferase II (GnTII) andα(1,6)-fucosyltransferase. In a specific embodiment the Fc domain of theimmunoconjugate is engineered to comprise an increased proportion ofnon-fucosylated oligosaccharides, as compared to a non-engineered Fcdomain, by producing the immunoconjugate in a host cell having increasedβ(1,4)-N-acetylglucosaminyltransferase III (GnTIII) activity. In an evenmore specific embodiment the host cell additionally has increasedα-mannosidase II (ManII) activity. The glycoengineering methodology thatcan be used for glycoengineering immunoconjugates of the presentinvention has been described in greater detail in Umana et al., NatBiotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93,851-861 (2006); WO 99/54342 (U.S. Pat. No. 6,602,684; EP 1071700); WO2004/065540 (U.S. Pat. Appl. Publ. No. 2004/0241817; EP 1587921), WO03/011878 (U.S. Pat. Appl. Publ. No. 2003/0175884), the content of eachof which is expressly incorporated herein by reference in its entirety.

Generally, any type of cultured cell line, including the cell linesdiscussed herein, can be used to generate cell lines for the productionof immunoconjugates with altered glycosylation pattern. Particular celllines include CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myelomacells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridomacells, and other mammalian cells. In certain embodiments, the host cellshave been manipulated to express increased levels of one or morepolypeptides having β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)activity. In certain embodiments the host cells have been furthermanipulated to express increased levels of one or more polypeptideshaving α-mannosidase II (ManII) activity. In a specific embodiment, thepolypeptide having GnTIII activity is a fusion polypeptide comprisingthe catalytic domain of GnTIII and the Golgi localization domain of aheterologous Golgi resident polypeptide. Particularly, said Golgilocalization domain is the Golgi localization domain of mannosidase II.Methods for generating such fusion polypeptides and using them toproduce antibodies with increased effector functions are disclosed inFerrara et al., Biotechn Bioeng 93, 851-861 (2006) and WO 2004/065540,the entire contents of which are expressly incorporated herein byreference.

The host cells which contain a coding sequence of an immunoconjugate ofthe invention and/or a coding sequence of a polypeptide havingglycosyltransferase activity, and which express the biologically activegene products, may be identified e.g. by DNA-DNA or DNA-RNAhybridization, the presence or absence of “marker” gene functions,assessing the level of transcription as measured by the expression ofthe respective mRNA transcripts in the host cell, or detection of thegene product as measured by immunoassay or by its biologicalactivity—methods which are well known in the art. GnTIII or Man IIactivity can be detected e.g. by employing a lectin which binds tobiosynthesis products of GnTIII or ManII, respectively. An example forsuch a lectin is the E₄-PHA lectin which binds preferentially tooligosaccharides containing bisecting GlcNAc. Biosynthesis products(i.e. specific oligosaccharide structures) of polypeptides having GnTIIIor ManII activity can also be detected by mass spectrometric analysis ofoligosaccharides released from glycoproteins produced by cellsexpressing said polypeptides. Alternatively, a functional assay whichmeasures the increased effector function and/or increased Fc receptorbinding, mediated by immunoconjugates produced by the cells engineeredwith the polypeptide having GnTIII or ManII activity may be used.

In another embodiment the Fc domain is engineered to comprise anincreased proportion of non-fucosylated oligosaccharides, as compared toa non-engineered Fc domain, by producing the immunoconjugate in a hostcell having decreased α(1,6)-fucosyltransferase activity. A host cellhaving decreased α(1,6)-fucosyltransferase activity may be a cell inwhich the α(1,6)-fucosyltransferase gene has been disrupted or otherwisedeactivated, e.g. knocked out (see Yamane-Ohnuki et al., Biotech Bioeng87, 614 (2004); Kanda et al., Biotechnol Bioeng 94(4), 680-688 (2006);Niwa et al., J Immunol Methods 306, 151-160 (2006)).

Other examples of cell lines capable of producing defucosylatedimmunoconjugates include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al., Arch Biochem Biophys 249, 533-545 (1986); USPat. Appl. No. US 2003/0157108; and WO 2004/056312, especially atExample 11). The immunoconjugates of the present invention canalternatively be glycoengineered to have reduced fucose residues in theFc domain according to the techniques disclosed in EP 1 176 195 A1, WO03/084570, WO 03/085119 and U.S. Pat. Appl. Pub. Nos. 2003/0115614,2004/093621, 2004/110282, 2004/110704, 2004/132140, U.S. Pat. No.6,946,292 (Kyowa), e.g. by reducing or abolishing the activity of aGDP-fucose transporter protein in the host cells used forimmunoconjugate production.

Glycoengineered immunoconjugates of the invention may also be producedin expression systems that produce modified glycoproteins, such as thosetaught in WO 2003/056914 (GlycoFi, Inc.) or in WO 2004/057002 and WO2004/024927 (Greenovation).

In one embodiment the Fc domain of the immunoconjugate is engineered tohave increased effector function, compared to a non-engineered Fcdomain. The increased effector function can include, but is not limitedto, one or more of the following: increased complement dependentcytotoxicity (CDC), increased antibody-dependent cell-mediatedcytotoxicity (ADCC), increased antibody-dependent cellular phagocytosis(ADCP), increased cytokine secretion, increased immune complex-mediatedantigen uptake by antigen-presenting cells, increased binding to NKcells, increased binding to macrophages, increased binding to monocytes,increased binding to polymorphonuclear cells, increased direct signalinginducing apoptosis, increased crosslinking of target-bound antibodies,increased dendritic cell maturation, or increased T cell priming.

In one embodiment the increased effector function is one or moreselected from the group of increased CDC, increased ADCC, increasedADCP, and increased cytokine secretion. In a particular embodiment theincreased effector function is increased ADCC. In one embodiment ADCCinduced by an engineered Fc domain (or an immunoconjugate comprising anengineered Fc domain) is a least 2-fold increased as compared to ADCCinduced by a non-engineered Fc domain (or an immunoconjugate comprisinga non-engineered Fc domain).

Effector Moieties

The effector moieties for use in the invention are generallypolypeptides that influence cellular activity, for example, throughsignal transduction pathways. Accordingly, the effector moiety of theimmunoconjugate useful in the invention can be associated withreceptor-mediated signaling that transmits a signal from outside thecell membrane to modulate a response within the cell. For example, aneffector moiety of the immunoconjugate can be a cytokine. In particularembodiments the effector moiety is human.

In certain embodiments the effector moiety is a single chain effectormoiety. In a particular embodiment the effector moiety is a cytokine.Examples of useful cytokines include, but are not limited to, GM-CSF,IL-la, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12,IL-21, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNF-α, and TNF-β. Inone embodiment the effector moiety of the immunoconjugate is a cytokineselected from the group of GM-CSF, IL-2, IL-7, IL-8, IL-10, IL-12,IL-15, IL-21, IFN-α, IFN-γ, MIP-1α, MIP-1β and TGF-β. In one embodimentthe effector moiety of the immunoconjugate is a cytokine selected fromthe group of IL-2, IL-7, IL-10, IL-12, IL-15, IFN-α, and IFN-γ. Incertain embodiments the cytokine effector moiety is mutated to remove N-and/or O-glycosylation sites. Elimination of glycosylation increaseshomogeneity of the product obtainable in recombinant production.

In a particular embodiment the effector moiety of the immunoconjugate isIL-2. In a specific embodiment, the IL-2 effector moiety can elicit oneor more of the cellular responses selected from the group consisting of:proliferation in an activated T lymphocyte cell, differentiation in anactivated T lymphocyte cell, cytotoxic T cell (CTL) activity,proliferation in an activated B cell, differentiation in an activated Bcell, proliferation in a natural killer (NK) cell, differentiation in aNK cell, cytokine secretion by an activated T cell or an NK cell, andNK/lymphocyte activated killer (LAK) antitumor cytotoxicity. In anotherparticular embodiment the IL-2 effector moiety is a mutant IL-2 effectormoiety having reduced binding affinity to the α-subunit of the IL-2receptor. Together with the β- and γ-subunits (also known as CD122 andCD132, respectively), the α-subunit (also known as CD25) forms theheterotrimeric high-affinity IL-2 receptor, while the dimeric receptorconsisting only of the β- and γ-subunits is termed theintermediate-affinity IL-2 receptor. As described in PCT patentapplication number PCT/EP2012/051991, which is incorporated herein byreference in its entirety, a mutant IL-2 polypeptide with reducedbinding to the α-subunit of the IL-2 receptor has a reduced ability toinduce IL-2 signaling in regulatory T cells, induces lessactivation-induced cell death (AICD) in T cells, and has a reducedtoxicity profile in vivo, compared to a wild-type IL-2 polypeptide. Theuse of such an effector moiety with reduced toxicity is particularlyadvantageous in an immunoconjugate according to the invention, having along serum half-life due to the presence of an Fc domain. In oneembodiment, the mutant IL-2 effector moiety of the immunoconjugateaccording to the invention comprises at least one amino acid mutationthat reduces or abolishes the affinity of the mutant IL-2 effectormoiety to the α-subunit of the IL-2 receptor (CD25) but preserves theaffinity of the mutant IL-2 effector moiety to the intermediate-affinityIL-2 receptor (consisting of the β- and γ-subunits of the IL-2receptor), compared to the non-mutated IL-2 effector moiety. In oneembodiment the one or more amino acid mutations are amino acidsubstitutions. In a specific embodiment, the mutant IL-2 effector moietycomprises one, two or three amino acid substitutions at one, two orthree position(s) selected from the positions corresponding to residue42, 45, and 72 of human IL-2. In a more specific embodiment, the mutantIL-2 effector moiety comprises three amino acid substitutions at thepositions corresponding to residue 42, 45 and 72 of human IL-2. In aneven more specific embodiment, the mutant IL-2 effector moiety is humanIL-2 comprising the amino acid substitutions F42A, Y45A and L72G. In oneembodiment the mutant IL-2 effector moiety additionally comprises anamino acid mutation at a position corresponding to position 3 of humanIL-2, which eliminates the O-glycosylation site of IL-2. Particularly,said additional amino acid mutation is an amino acid substitutionreplacing a threonine residue by an alanine residue. A particular mutantIL-2 effector moiety useful in the invention comprises four amino acidsubstitutions at positions corresponding to residues 3, 42, 45 and 72 ofhuman IL-2. Specific amino acid substitutions are T3A, F42A, Y45A andL72G. As demonstrated in PCT patent application number PCT/EP2012/051991and in the appended Examples, said quadruple mutant IL-2 polypeptide(IL-2 qm) exhibits no detectable binding to CD25, reduced ability toinduce apoptosis in T cells, reduced ability to induce IL-2 signaling inT_(reg) cells, and a reduced toxicity profile in vivo. However, itretains ability to activate IL-2 signaling in effector cells, to induceproliferation of effector cells, and to generate IFN-γ as a secondarycytokine by NK cells.

The IL-2 or mutant IL-2 effector moiety according to any of the aboveembodiments may comprise additional mutations that provide furtheradvantages such as increased expression or stability. For example, thecysteine at position 125 may be replaced with a neutral amino acid suchas alanine, to avoid the formation of disulfide-bridged IL-2 dimers.Thus, in certain embodiments the IL-2 or mutant IL-2 effector moiety ofthe immunoconjugate according to the invention comprises an additionalamino acid mutation at a position corresponding to residue 125 of humanIL-2. In one embodiment said additional amino acid mutation is the aminoacid substitution C125A.

In a specific embodiment the IL-2 effector moiety of the immunoconjugatecomprises the polypeptide sequence of SEQ ID NO: 2. In another specificembodiment the IL-2 effector moiety of the immunoconjugate comprises thepolypeptide sequence of SEQ ID NO: 3.

In another embodiment the effector moiety of the immunoconjugate isIL-12. In a specific embodiment said IL-12 effector moiety is a singlechain IL-12 effector moiety. In an even more specific embodiment thesingle chain IL-12 effector moiety comprises the polypeptide sequence ofSEQ ID NO: 4. In one embodiment, the IL-12 effector moiety can elicitone or more of the cellular responses selected from the group consistingof: proliferation in a NK cell, differentiation in a NK cell,proliferation in a T cell, and differentiation in a T cell.

In another embodiment the effector moiety of the immunoconjugate isIL-10. In a specific embodiment said IL-10 effector moiety is a singlechain IL-10 effector moiety. In an even more specific embodiment thesingle chain IL-10 effector moiety comprises the polypeptide sequence ofSEQ ID NO: 5. In another specific embodiment the IL-10 effector moietyis a monomeric IL-10 effector moiety. In a more specific embodiment themonomeric IL-10 effector moiety comprises the polypeptide sequence ofSEQ ID NO: 6. In one embodiment, the IL-10 effector moiety can elicitone or more of the cellular responses selected from the group consistingof: inhibition of cytokine secretion, inhibition of antigen presentationby antigen presenting cells, reduction of oxygen radical release, andinhibition of T cell proliferation. An immunoconjugate according to theinvention wherein the effector moiety is IL-10 is particularly usefulfor downregulation of inflammation, e.g. in the treatment of aninflammatory disorder.

In another embodiment the effector moiety of the immunoconjugate isIL-15. In a specific embodiment said IL-15 effector moiety is a mutantIL-15 effector moiety having reduced binding affinity to the α-subunitof the IL-15 receptor. Without wishing to be bound by theory, a mutantIL-15 polypeptide with reduced binding to to the α-subunit of the IL-15receptor has a reduced ability to bind to fibroblasts throughout thebody, resulting in improved pharmacokinetics and toxicity profile,compared to a wild-type IL-15 polypeptide. The use of an effector moietywith reduced toxicity, such as the described mutant IL-2 and mutantIL-15 effector moieties, is particularly advantageous in animmunoconjugate according to the invention, having a long serumhalf-life due to the presence of an Fc domain. In one embodiment themutant IL-15 effector moiety of the immunoconjugate according to theinvention comprises at least one amino acid mutation that reduces orabolishes the affinity of the mutant IL-15 effector moiety to theα-subunit of the IL-15 receptor but preserves the affinity of the mutantIL-15 effector moiety to the intermediate-affinity IL-15/IL-2 receptor(consisting of the β- and γ-subunits of the IL-15/IL-2 receptor),compared to the non-mutated IL-15 effector moiety. In one embodiment theamino acid mutation is an amino acid substitution. In a specificembodiment, the mutant IL-15 effector moiety comprises an amino acidsubstitution at the position corresponding to residue 53 of human IL-15.In a more specific embodiment, the mutant IL-15 effector moiety is humanIL-15 comprising the amino acid substitution E53A. In one embodiment themutant IL-15 effector moiety additionally comprises an amino acidmutation at a position corresponding to position 79 of human IL-15,which eliminates the N-glycosylation site of IL-15. Particularly, saidadditional amino acid mutation is an amino acid substitution replacingan asparagine residue by an alanine residue. In an even more specificembodiment the IL-15 effector moiety comprises the polypeptide sequenceof SEQ ID NO: 7. In one embodiment, the IL-15 effector moiety can elicitone or more of the cellular responses selected from the group consistingof: proliferation in an activated T lymphocyte cell, differentiation inan activated T lymphocyte cell, cytotoxic T cell (CTL) activity,proliferation in an activated B cell, differentiation in an activated Bcell, proliferation in a natural killer (NK) cell, differentiation in aNK cell, cytokine secretion by an activated T cell or an NK cell, andNK/lymphocyte activated killer (LAK) antitumor cytotoxicity.

Mutant cytokine molecules useful as effector moieties in theimmunoconjugates can be prepared by deletion, substitution, insertion ormodification using genetic or chemical methods well known in the art.Genetic methods may include site-specific mutagenesis of the encodingDNA sequence, PCR, gene synthesis, and the like. The correct nucleotidechanges can be verified for example by sequencing. Substitution orinsertion may involve natural as well as non-natural amino acidresidues. Amino acid modification includes well known methods ofchemical modification such as the addition or removal of glycosylationsites or carbohydrate attachments, and the like.

In one embodiment, the effector moiety, particularly a single-chaineffector moiety, of the immunoconjugate is GM-CSF. In a specificembodiment, the GM-CSF effector moiety can elicit proliferation and/ordifferentiation in a granulocyte, a monocyte or a dendritic cell. In oneembodiment, the effector moiety, particularly a single-chain effectormoiety, of the immunoconjugate is IFN-α. In a specific embodiment, theIFN-α effector moiety can elicit one or more of the cellular responsesselected from the group consisting of: inhibiting viral replication in avirus-infected cell, and upregulating the expression of majorhistocompatibility complex I (MHC I). In another specific embodiment,the IFN-α effector moiety can inhibit proliferation in a tumor cell. Inone embodiment the effector moiety, particularly a single-chain effectormoiety, of the immunoconjugate is IFN-γ. In a specific embodiment, theIFN-γ effector moiety can elicit one or more of the cellular responsesselected from the group of: increased macrophage activity, increasedexpression of MHC molecules, and increased NK cell activity. In oneembodiment the effector moiety, particularly a single-chain effectormoiety, of the immunoconjugate is IL-7. In a specific embodiment, theIL-7 effector moiety can elicit proliferation of T and/or B lymphocytes.In one embodiment, the effector moiety, particularly a single-chaineffector moiety, of the immunoconjugate is IL-8. In a specificembodiment, the IL-8 effector moiety can elicit chemotaxis inneutrophils. In one embodiment, the effector moiety, particularly asingle-chain effector moiety, of the immunoconjugate, is MIP-1α. In aspecific embodiment, the MIP-la effector moiety can elicit chemotaxis inmonocytes and T lymphocyte cells. In one embodiment, the effectormoiety, particularly a single-chain effector moiety, of theimmunoconjugate is MIP-1β. In a specific embodiment, the MIP-1β effectormoiety can elicit chemotaxis in monocytes and T lymphocyte cells. In oneembodiment, the effector moiety, particularly a single-chain effectormoiety, of the immunoconjugate is TGF-β. In a specific embodiment, theTGF-β effector moiety can elicit one or more of the cellular responsesselected from the group consisting of: chemotaxis in monocytes,chemotaxis in macrophages, upregulation of IL-1 expression in activatedmacrophages, and upregulation of IgA expression in activated B cells.

In one embodiment, the immunoconjugate of the invention binds to aneffector moiety receptor with a dissociation constant (K_(D)) that is atleast about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8,8.5, 9, 9.5 or 10 times greater than that for a control effector moiety.In another embodiment, the immunoconjugate binds to an effector moietyreceptor with a K_(D) that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10times greater than that for a corresponding immunoconjugate moleculecomprising two or more effector moieties. In another embodiment, theimmunoconjugate binds to an effector moiety receptor with a dissociationconstant K_(D) that is about 10 times greater than that for acorresponding immunoconjugate molecule comprising two or more effectormoieties.

Antigen Binding Moieties

The immunoconjugates of the invention comprise at least one antigenbinding moiety. In particular embodiments, the immunoconjugatescomprises two antigen binding moieties, i.e. a first and a secondantigen binding moiety. In one embodiment the immunoconjugate comprisesnot more than two antigen binding moieties.

The antigen binding moiety of the immunoconjugate of the invention isgenerally a polypeptide molecule that binds to a specific antigenicdeterminant and is able to direct the entity to which it is attached(e.g. an effector moiety and an Fc domain) to a target site, for exampleto a specific type of tumor cell or tumor stroma that bears theantigenic determinant. The immunoconjugate can bind to antigenicdeterminants found, for example, on the surfaces of tumor cells, on thesurfaces of virus-infected cells, on the surfaces of other diseasedcells, free in blood serum, and/or in the extracellular matrix (ECM).

In certain embodiments the antigen binding moiety is directed to anantigen associated with a pathological condition, such as an antigenpresented on a tumor cell or in a tumor cell environment, at a site ofinflammation, or on a virus-infected cell.

Non-limiting examples of tumor antigens include MAGE, MART-1/Melan-A,gp100, Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-bindingprotein (ADAbp), cyclophilin b, Colorectal associated antigen(CRC)-0017-1A/GA733, Carcinoembryonic Antigen (CEA) and its immunogenicepitopes CAP-1 and CAP-2, etv6, aml1, Prostate Specific Antigen (PSA)and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specificmembrane antigen (PSMA), MAGE-family of tumor antigens (e.g., MAGE-A1,MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9,MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3),MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-05),GAGE-family of tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4,GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V,MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21 ras, RCAS1,α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn,gp100 Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coliprotein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2gangliosides, viral products such as human papilloma virus proteins,Smad family of tumor antigens, lmp-1, NA, EBV-encoded nuclear antigen(EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40),SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2.

Non-limiting examples of viral antigens include influenza virushemagglutinin, Epstein-Barr virus LMP-1, hepatitis C virus E2glycoprotein, HIV gp160, and HIV gp120.

Non-limiting examples of ECM antigens include syndecan, heparanase,integrins, osteopontin, link, cadherins, laminin, laminin type EGF,lectin, fibronectin, notch, tenascin, and matrixin.

The immunoconjugates of the invention can bind to the following specificnon-limiting examples of cell surface antigens: FAP, Her2, EGFR, IGF-1R,CD22 (B-cell receptor), CD23 (low affinity IgE receptor), CD30 (cytokinereceptor), CD33 (myeloid cell surface antigen), CD40 (tumor necrosisfactor receptor), IL-6R (IL6 receptor), CD20, MCSP, and PDGFβR (βplatelet-derived growth factor receptor). In particular embodiments theantigen is a human antigen.

In certain embodiments the antigen-binding moiety is directed to anantigen presented on a tumor cell or in a tumor cell environment. Inother embodiments the antigen binding moiety is directed to an antigenpresented at a site of inflammation. In a specific embodiment theantigen-binding moiety is directed to an antigen selected from the groupof Fibroblast Activation Protein (FAP), the A1 domain of Tenascin-C (TNCA1), the A2 domain of Tenascin-C (TNC A2), the Extra Domain B ofFibronectin (EDB), Carcinoembryonic Antigen (CEA), andMelanoma-associated Chondroitin Sulfate Proteoglycan (MCSP).

In one embodiment, the immunoconjugate of the invention comprises two ormore antigen binding moieties, wherein each of these antigen bindingmoieties specifically binds to the same antigenic determinant.

The antigen binding moiety can be any type of antibody or fragmentthereof that retains specific binding to an antigenic determinant.Antibody fragments include, but are not limited to, V_(H) fragments,V_(L) fragments, Fab fragments, F(ab)₂ fragments, scFv fragments, F_(V)fragments, minibodies, diabodies, triabodies, and tetrabodies (see e.g.Hudson and Souriau, Nature Med 9, 129-134 (2003)). In a particularembodiment the antigen binding moiety is a Fab molecule. In oneembodiment said Fab molecule is human. In another embodiment said Fabmolecule is humanized. In yet another embodiment said Fab moleculecomprises human heavy and light chain constant regions.

In one embodiment the immunoconjugate comprises at least one, typicallytwo or more antigen binding moieties that are specific for the ExtraDomain B of fibronectin (EDB). In another embodiment the immunoconjugatecomprises at least one, typically two or more antigen binding moietiesthat can compete with monoclonal antibody L19 for binding to an epitopeof EDB. See, e.g., PCT publication WO 2007/128563 A1 (incorporatedherein by reference in its entirety).

In yet another embodiment the immunoconjugate comprises a polypeptidesequence wherein a Fab heavy chain derived from the L19 monoclonalantibody shares a carboxy-terminal peptide bond with an Fc domainsubunit comprising a knob modification, which in turn shares acarboxy-terminal peptide bond with an IL-2 polypeptide. In a morespecific embodiment the immunoconjugate comprises the polypeptidesequence of SEQ ID NO: 215 or a variant thereof that retainsfunctionality. In one embodiment the immunoconjugate comprises apolypeptide sequence wherein a Fab heavy chain derived from the L19monoclonal antibody shares a carboxy-terminal peptide bond with an Fcdomain subunit comprising a hole modification. In a more specificembodiment the immunoconjugate comprises the polypeptide sequence of SEQID NO: 213 or a variant thereof that retains functionality. In anotherembodiment the immunoconjugate comprises a Fab light chain derived fromthe L19 monoclonal antibody. In a more specific embodiment theimmunoconjugate comprises the polypeptide sequence of SEQ ID NO: 217 ora variant thereof that retains functionality. In yet another embodimentthe immunoconjugate comprises the polypeptide sequences of SEQ ID NO:213, SEQ ID NO: 215 and SEQ ID NO: 217, or variants thereof that retainfunctionality. In another specific embodiment the polypeptides arecovalently linked, e.g., by a disulfide bond. In some embodiments the Fcdomain subunits each comprise the amino acid substitutions L234A, L235A,and P329G.

In a specific embodiment the immunoconjugate comprises a polypeptidesequence encoded by a polynucleotide sequence that is at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence ofSEQ ID NO: 216. In another specific embodiment the immunoconjugatecomprises a polypeptide sequence encoded by the polynucleotide sequenceof SEQ ID NO: 216. In another specific embodiment the immunoconjugatecomprises a polypeptide sequence encoded by a polynucleotide sequencethat is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 214. In yet another specificembodiment the immunoconjugate comprises a polypeptide sequence encodedby the polynucleotide sequence of SEQ ID NO: 214. In another specificembodiment the immunoconjugate comprises a polypeptide sequence encodedby a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 218. Inyet another specific embodiment the immunoconjugate comprises apolypeptide sequence encoded by the polynucleotide sequence of SEQ IDNO: 218.

In one embodiment the immunoconjugate of the invention comprises atleast one, typically two or more antigen binding moieties that arespecific for the A1 domain of Tenascin C (TNC-A1).

In another embodiment, the immunoconjugate comprises at least one,typically two or more antigen binding moieties that can compete withmonoclonal antibody F16 for binding to an epitope of TNC-A1. See, e.g.,PCT publication WO 2007/128563 A1 (incorporated herein by reference inits entirety). In one embodiment, the immunoconjugate comprises at leastone, typically two or more antigen binding moieties that are specificfor the A1 and/or the A4 domain of Tenascin C (TNC-A1 or TNC-A4 orTNC-A1/A4).

In a specific embodiment, the antigen binding moieties of theimmunoconjugate comprise a heavy chain variable region sequence that isat least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identicalto either SEQ ID NO: 33 or SEQ ID NO: 35, or variants thereof thatretain functionality. In another specific embodiment, the antigenbinding moieties of the immunoconjugate comprise a light chain variableregion sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% identical to either SEQ ID NO: 29 or SEQ ID NO: 31, orvariants thereof that retain functionality. In a more specificembodiment, the antigen binding moieties of the immunoconjugate comprisea heavy chain variable region sequence that is at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identical to either SEQ ID NO: 33or SEQ ID NO: 35 or variants thereof that retain functionality, and alight chain variable region sequence that is at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identical to either SEQ ID NO: 29or SEQ ID NO: 31 or variants thereof that retain functionality. Inanother specific embodiment, the heavy chain variable region sequence ofthe antigen binding moieties of the immunoconjugate is encoded by apolynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to either SEQ ID NO: 34 or SEQ ID NO: 36. Inyet another specific embodiment, the heavy chain variable regionsequence of the antigen binding moieties of the immunoconjugate isencoded by the polynucleotide sequence of either SEQ ID NO: 34 or SEQ IDNO: 36. In another specific embodiment, the light chain variable regionsequence of the antigen binding moieties of the immunoconjugate isencoded by a polynucleotide sequence that is at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to either SEQ ID NO: 30 or SEQID NO: 32. In yet another specific embodiment, the light chain variableregion sequence of the antigen binding moieties of the immunoconjugateis encoded by the polynucleotide sequence of either SEQ ID NO: 30 or SEQID NO: 32.

In one embodiment, the immunoconjugate comprises a polypeptide sequencewherein a Fab heavy chain specific for the A1 domain of Tenascin Cshares a carboxy-terminal peptide bond with an Fc domain subunitcomprising a knob modification, which in turn shares a carboxy-terminalpeptide bond with an IL-2 polypeptide. In another embodiment, theimmunoconjugate comprises a polypeptide sequence wherein a Fab heavychain specific for the A1 domain of Tenascin C shares a carboxy-terminalpeptide bond with an Fc domain subunit comprising a hole modification.In a more specific embodiment the immunoconjugate comprises both ofthese polypeptide sequences. In another embodiment, the immunoconjugatefurther comprises a Fab light chain specific for the A1 domain ofTenascin C. In another specific embodiment, the polypeptides arecovalently linked, e.g., by a disulfide bond. In some embodiments the Fcdomain subunits each comprise the amino acid substitutions L234A, L235A,and P329G.

In a particular embodiment, the immunoconjugate comprises at least one,typically two or more antigen binding moieties that are specific for theA2 domain of Tenascin C (TNC-A2). In a specific embodiment, the antigenbinding moieties of the immunoconjugate comprise a heavy chain variableregion sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% identical to a sequence selected from the group of SEQID NO: 27, SEQ ID NO: 159, SEQ ID NO: 163, SEQ ID NO: 167, SEQ ID NO:171, SEQ ID NO:175, SEQ ID NO: 179, SEQ ID NO: 183 and SEQ ID NO: 187,or variants thereof that retain functionality. In another specificembodiment, the antigen binding moieties of the immunoconjugate comprisea light chain variable region sequence that is at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selectedfrom the group of SEQ ID NO: 23, SEQ ID NO: 25; SEQ ID NO: 157, SEQ IDNO: 161, SEQ ID NO:165, SEQ ID NO: 169, SEQ ID NO: 173, SEQ ID NO: 177,SEQ ID NO: 181 and SEQ ID NO: 185, or variants thereof that retainfunctionality. In a more specific embodiment, the antigen bindingmoieties of the immunoconjugate comprise a heavy chain variable regionsequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to a sequence selected from the group of SEQ ID NO:27, SEQ ID NO: 159, SEQ ID NO: 163, SEQ ID NO: 167, SEQ ID NO: 171, SEQID NO:175, SEQ ID NO: 179, SEQ ID NO: 183 and SEQ ID NO: 187, orvariants thereof that retain functionality, and a light chain variableregion sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% identical to a sequence selected from the group of SEQID NO: 23, SEQ ID NO: 25; SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO:165,SEQ ID NO: 169, SEQ ID NO: 173, SEQ ID NO: 177, SEQ ID NO: 181 and SEQID NO: 185, or variants thereof that retain functionality. In aparticular embodiment, the antigen binding moieties of theimmunoconjugate comprise the heavy chain variable region sequence of SEQID NO: 27 and the light chain variable region sequence of SEQ ID NO: 25.

In another specific embodiment, the heavy chain variable region sequenceof the antigen binding moieties of the immunoconjugate is encoded by apolynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to a sequence selected from the group of SEQID NO: 28, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO: 168, SEQ ID NO:172, SEQ ID NO: 176, SEQ ID NO: 180, SEQ ID NO: 184 and SEQ ID NO: 188.In yet another specific embodiment, the heavy chain variable regionsequence of the antigen binding moieties of the immunoconjugate isencoded by a polynucleotide sequence selected from the group of SEQ IDNO: 28, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO: 168, SEQ ID NO: 172,SEQ ID NO: 176, SEQ ID NO: 180, SEQ ID NO: 184 and SEQ ID NO: 188. Inanother specific embodiment, the light chain variable region sequence ofthe antigen binding moieties of the immunoconjugate is encoded by apolynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to a sequence selected from the group of SEQID NO: 24, SEQ ID NO: 26, SEQ ID NO: 158, SEQ ID NO: 162, SEQ ID NO:166, SEQ ID NO: 170, SEQ ID NO: 174, SEQ ID NO: 178, SEQ ID NO: 182 andSEQ ID NO: 186. In yet another specific embodiment, the light chainvariable region sequence of the antigen binding moieties of theimmunoconjugate is encoded by a polynucleotide sequence selected fromthe group of SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 158, SEQ ID NO:162, SEQ ID NO: 166, SEQ ID NO: 170, SEQ ID NO: 174, SEQ ID NO: 178, SEQID NO: 182 and SEQ ID NO: 186.

In yet another embodiment, the immunoconjugate comprises a polypeptidesequence wherein a Fab heavy chain specific for the A2 domain ofTenascin C shares a carboxy-terminal peptide bond with an Fc domainsubunit comprising a hole modification, which in turn shares acarboxy-terminal peptide bond with an IL-10 polypeptide. In a morespecific embodiment, the immunoconjugate comprises the polypeptidesequence of SEQ ID NO: 235 or SEQ ID NO: 237, or a variant thereof thatretains functionality. In one embodiment the immunoconjugate comprises apolypeptide sequence wherein a Fab heavy chain specific for the A2domain of Tenascin C shares a carboxy-terminal peptide bond with an Fcdomain subunit comprising a knob modification. In a more specificembodiment, the immunoconjugate comprises the polypeptide sequence ofSEQ ID NO: 233 or a variant thereof that retains functionality. Inanother embodiment, the immunoconjugate comprises a Fab light chainspecific for the A2 domain of Tenascin C. In a more specific embodiment,the immunoconjugate comprises the polypeptide sequence of SEQ ID NO: 239or a variant thereof that retains functionality. In another embodiment,the immunoconjugate comprises the polypeptide sequences of SEQ ID NO:233, SEQ ID NO: 235 and SEQ ID NO: 239 or variants thereof that retainfunctionality. In yet another embodiment, the immunoconjugate comprisesthe polypeptide sequences of SEQ ID NO: 233, SEQ ID NO: 237 and SEQ IDNO: 239 or variants thereof that retain functionality. In anotherspecific embodiment, the polypeptides are covalently linked, e.g., by adisulfide bond. In some embodiments the Fc domain subunits each comprisethe amino acid substitutions L234A, L235A, and P329G.

In a specific embodiment, the immunoconjugate comprises a polypeptidesequence encoded by a polynucleotide sequence that is at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence ofSEQ ID NO: 236 or SEQ ID NO: 238. In another specific embodiment, theimmunoconjugate comprises a polypeptide sequence encoded by thepolynucleotide sequence of SEQ ID NO: 236 or SEQ ID NO: 238. In anotherspecific embodiment, the immunoconjugate comprises a polypeptidesequence encoded by a polynucleotide sequence that is at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence ofSEQ ID NO: 234. In yet another specific embodiment, the immunoconjugatecomprises a polypeptide sequence encoded by the polynucleotide sequenceof SEQ ID NO: 234. In another specific embodiment, the immunoconjugatecomprises a polypeptide sequence encoded by a polynucleotide sequencethat is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 240. In yet another specificembodiment, the immunoconjugate comprises a polypeptide sequence encodedby the polynucleotide sequence of SEQ ID NO: 240.

In yet another embodiment, the immunoconjugate comprises a polypeptidesequence wherein a Fab heavy chain specific for the A2 domain ofTenascin C shares a carboxy-terminal peptide bond with an Fc domainsubunit comprising a knob modification, which in turn shares acarboxy-terminal peptide bond with an IL-2 polypeptide. In a morespecific embodiment, the immunoconjugate comprises the polypeptidesequence of SEQ ID NO: 285, or a variant thereof that retainsfunctionality. In one embodiment the immunoconjugate comprises apolypeptide sequence wherein a Fab heavy chain specific for the A2domain of Tenascin C shares a carboxy-terminal peptide bond with an Fcdomain subunit comprising a hole modification. In a more specificembodiment, the immunoconjugate comprises the polypeptide sequence ofSEQ ID NO: 287, or a variant thereof that retains functionality. Inanother embodiment, the immunoconjugate comprises a Fab light chainspecific for the A2 domain of Tenascin C. In a more specific embodiment,the immunoconjugate comprises the polypeptide sequence of SEQ ID NO: 239or a variant thereof that retains functionality. In another embodiment,the immunoconjugate comprises the polypeptide sequences of SEQ ID NO:285, SEQ ID NO: 287 and SEQ ID NO: 239 or variants thereof that retainfunctionality. In another specific embodiment, the polypeptides arecovalently linked, e.g., by a disulfide bond. In some embodiments the Fcdomain subunits each comprise the amino acid substitutions L234A, L235A,and P329G.

In a specific embodiment, the immunoconjugate comprises a polypeptidesequence encoded by a polynucleotide sequence that is at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence ofSEQ ID NO: 286. In another specific embodiment, the immunoconjugatecomprises a polypeptide sequence encoded by the polynucleotide sequenceof SEQ ID NO: 286. In another specific embodiment, the immunoconjugatecomprises a polypeptide sequence encoded by a polynucleotide sequencethat is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 288. In yet another specificembodiment, the immunoconjugate comprises a polypeptide sequence encodedby the polynucleotide sequence of SEQ ID NO: 288. In another specificembodiment, the immunoconjugate comprises a polypeptide sequence encodedby a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 240. Inyet another specific embodiment, the immunoconjugate comprises apolypeptide sequence encoded by the polynucleotide sequence of SEQ IDNO: 240.

In a particular embodiment, the immunoconjugate comprises at least one,typically two or more antigen binding moieties that are specific for theFibroblast Activated Protein (FAP). In a specific embodiment, theantigen binding moieties of the immunoconjugate comprise a heavy chainvariable region sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to a sequence selected from the groupconsisting of SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO:51, SEQ ID NO: 55, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 67, SEQ IDNO: 71, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQID NO: 91, SEQ ID NO: 95, SEQ ID NO: 99, SEQ ID NO: 103, SEQ ID NO: 107,SEQ ID NO: 111, SEQ ID NO: 115, SEQ ID NO: 119, SEQ ID NO: 123, SEQ IDNO: 127, SEQ ID NO: 131, SEQ ID NO: 135, SEQ ID NO: 139, SEQ ID NO: 143,SEQ ID NO: 147, SEQ ID NO: 151 and SEQ ID NO: 155, or variants thereofthat retain functionality. In another specific embodiment, the antigenbinding moieties of the immunoconjugate comprise a light chain variableregion sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% identical to a sequence selected from the groupconsisting of: SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO:49, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: 65, SEQ IDNO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 85, SEQID NO: 89, SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO: 101, SEQ ID NO: 105,SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 117, SEQ ID NO: 121, SEQ IDNO: 125, SEQ ID NO: 129, SEQ ID NO: 133, SEQ ID NO: 137, SEQ ID NO: 141,SEQ ID NO: 145, SEQ ID NO: 149 and SEQ ID NO: 153, or variants thereofthat retain functionality. In a more specific embodiment, the antigenbinding moieties of the immunoconjugate comprise a heavy chain variableregion sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% identical to a sequence selected from the groupconsisting of SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO:51, SEQ ID NO: 55, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 67, SEQ IDNO: 71, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQID NO: 91, SEQ ID NO: 95, SEQ ID NO: 99, SEQ ID NO: 103, SEQ ID NO: 107,SEQ ID NO: 111, SEQ ID NO: 115, SEQ ID NO: 119, SEQ ID NO: 123, SEQ IDNO: 127, SEQ ID NO: 131, SEQ ID NO: 135, SEQ ID NO: 139, SEQ ID NO: 143,SEQ ID NO: 147, SEQ ID NO: 151 and SEQ ID NO: 155, or variants thereofthat retain functionality, and a light chain variable region sequencethat is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to a sequence selected from the group consisting of: SEQ IDNO: 37, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 49, SEQ ID NO: 53, SEQID NO: 57, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 73,SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO:93, SEQ ID NO: 97, SEQ ID NO: 101, SEQ ID NO: 105, SEQ ID NO: 109, SEQID NO: 113, SEQ ID NO: 117, SEQ ID NO: 121, SEQ ID NO: 125, SEQ ID NO:129, SEQ ID NO: 133, SEQ ID NO: 137, SEQ ID NO: 141, SEQ ID NO: 145, SEQID NO: 149 and SEQ ID NO: 153, or variants thereof that retainfunctionality. In a particular embodiment, the antigen binding moietiesof the immunoconjugate comprise the heavy chain variable region sequenceof SEQ ID NO: 111 and the light chain variable region sequence of SEQ IDNO: 109. In a further particular embodiment, the antigen bindingmoieties of the immunoconjugate comprise the heavy chain variable regionsequence of SEQ ID NO: 143 and the light chain variable region sequenceof SEQ ID NO: 141. In yet another particular embodiment, the antigenbinding moieties of the immunoconjugate comprise the heavy chainvariable region sequence of SEQ ID NO: 51 and the light chain variableregion sequence of SEQ ID NO: 49.

In another specific embodiment, the heavy chain variable region sequenceof the antigen binding moieties of the immunoconjugate is encoded by apolynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to a sequence selected from the groupconsisting of: SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO:52, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68, SEQ IDNO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 84, SEQ ID NO: 88, SEQID NO: 92, SEQ ID NO: 96, SEQ ID NO: 100, SEQ ID NO: 104, SEQ ID NO:108, SEQ ID NO: 112, SEQ ID NO: 116, SEQ ID NO: 120, SEQ ID NO: 124, SEQID NO: 128, SEQ ID NO: 132, SEQ ID NO: 136, SEQ ID NO: 140, SEQ ID NO:144, SEQ ID NO: 148, SEQ ID NO: 152, and SEQ ID NO: 156. In yet anotherspecific embodiment, the heavy chain variable region sequence of theantigen binding moieties of the immunoconjugate is encoded by apolynucleotide sequence selected from the group consisting of: SEQ IDNO: 42, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, SEQID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76,SEQ ID NO: 80, SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 92, SEQ ID NO:96, SEQ ID NO: 100, SEQ ID NO: 104, SEQ ID NO: 108, SEQ ID NO: 112, SEQID NO: 116, SEQ ID NO: 120, SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO:132, SEQ ID NO: 136, SEQ ID NO: 140, SEQ ID NO: 144, SEQ ID NO: 148, SEQID NO: 152, and SEQ ID NO: 156. In another specific embodiment, thelight chain variable region sequence of the antigen binding moieties ofthe immunoconjugate is encoded by a polynucleotide sequence that is atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical tosequence selected from the group consisting of: SEQ ID NO: 38, SEQ IDNO: 40, SEQ ID NO: 44, SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, SEQID NO: 62, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 74, SEQ ID NO: 78,SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 90, SEQ ID NO: 94, SEQ ID NO:98, SEQ ID NO: 102, SEQ ID NO: 106, SEQ ID NO: 110, SEQ ID NO: 114, SEQID NO: 118, SEQ ID NO: 122, SEQ ID NO: 126, SEQ ID NO: 130, SEQ ID NO:134, SEQ ID NO: 138, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 150, andSEQ ID NO: 154. In yet another specific embodiment, the light chainvariable region sequence of the antigen binding moieties of theimmunoconjugate is encoded by a polynucleotide sequence selected fromthe group consisting of: SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44,SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO:66, SEQ ID NO: 70, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ IDNO: 86, SEQ ID NO: 90, SEQ ID NO: 94, SEQ ID NO: 98, SEQ ID NO: 102, SEQID NO: 106, SEQ ID NO: 110, SEQ ID NO: 114, SEQ ID NO: 118, SEQ ID NO:122, SEQ ID NO: 126, SEQ ID NO: 130, SEQ ID NO: 134, SEQ ID NO: 138, SEQID NO: 142, SEQ ID NO: 146, SEQ ID NO: 150, and SEQ ID NO: 154.

In one embodiment, the immunoconjugate comprises a polypeptide sequencewherein a Fab heavy chain specific for FAP shares a carboxy-terminalpeptide bond with an Fc domain subunit comprising a knob modification,which in turn shares a carboxy-terminal peptide bond with an IL-2polypeptide. In a more specific embodiment, the immunoconjugatecomprises a polypeptide sequence selected from the group of SEQ ID NO:195, SEQ ID NO: 197, SEQ ID NO: 203, SEQ ID NO: 209, SEQ ID NO: 269, SEQID NO: 271 and SEQ ID NO: 273, or variants thereof that retainfunctionality. In one embodiment, the immunoconjugate comprises apolypeptide sequence wherein a Fab heavy chain specific for FAP shares acarboxy-terminal peptide bond with an Fc domain subunit comprising aknob modification, which in turn shares a carboxy-terminal peptide bondwith an IL-15 polypeptide. In a more specific embodiment, theimmunoconjugate comprises the polypeptide sequence of SEQ ID NO: 199, ora variant thereof that retains functionality. In one embodiment theimmunoconjugate comprises a polypeptide sequence wherein a Fab heavychain specific for FAP shares a carboxy-terminal peptide bond with an Fcdomain subunit comprising a hole modification. In a more specificembodiment, the immunoconjugate comprises a polypeptide sequenceselected from the group of SEQ ID NO: 193, SEQ ID NO: 201 and SEQ ID NO:207, or variants thereof that retain functionality. In anotherembodiment, the immunoconjugate comprises a Fab light chain specific forFAP. In a more specific embodiment, the immunoconjugate comprises thepolypeptide sequence of SEQ ID NO: 205 or SEQ ID NO: 211, or a variantthereof that retains functionality. In another embodiment, theimmunoconjugate comprises the polypeptide sequence of SEQ ID NO: 205,the polypeptide sequence of SEQ ID NO: 193, and a polypeptide sequenceselected from the group of SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO:199 and SEQ ID NO: 269, or variants thereof that retain functionality.In yet another embodiment, the immunoconjugate comprises the polypeptidesequences of SEQ ID NO: 201, SEQ ID NO: 203 and SEQ ID NO: 205, orvariants thereof that retain functionality. In yet another embodiment,the immunoconjugate comprises the polypeptide sequences of SEQ ID NO:207, SEQ ID NO: 209 and SEQ ID NO: 211, or variants thereof that retainfunctionality. In yet another embodiment, the immunoconjugate comprisesthe polypeptide sequences of SEQ ID NO: 205, SEQ ID NO: 193 and SEQ IDNO: 269, or variants thereof that retain functionality. In yet anotherembodiment, the immunoconjugate comprises the polypeptide sequences ofSEQ ID NO: 211, SEQ ID NO: 207 and SEQ ID NO: 271, or variants thereofthat retain functionality. In yet another embodiment, theimmunoconjugate comprises the polypeptide sequences of SEQ ID NO: 211,SEQ ID NO: 207 and SEQ ID NO: 273, or variants thereof that retainfunctionality. In another specific embodiment, the polypeptides arecovalently linked, e.g., by a disulfide bond. In some embodiments the Fcdomain subunits each comprise the amino acid substitutions L234A, L235A,and P329G.

In yet another embodiment, the immunoconjugate comprises a polypeptidesequence wherein a Fab heavy chain specific for FAP shares acarboxy-terminal peptide bond with an Fc domain subunit comprising ahole modification, which in turn shares a carboxy-terminal peptide bondwith an IL-10 polypeptide. In a more specific embodiment, theimmunoconjugate comprises the polypeptide sequence of SEQ ID NO: 243 orSEQ ID NO: 245, or a variant thereof that retains functionality. In oneembodiment the immunoconjugate comprises a polypeptide sequence whereina Fab heavy chain specific for FAP shares a carboxy-terminal peptidebond with an Fc domain subunit comprising a knob modification. In a morespecific embodiment, the immunoconjugate comprises the polypeptidesequence of SEQ ID NO: 241 or a variant thereof that retainsfunctionality. In another embodiment, the immunoconjugate comprises aFab light chain specific for FAP. In a more specific embodiment, theimmunoconjugate comprises the polypeptide sequence of SEQ ID NO: 205 ora variant thereof that retains functionality. In another embodiment, theimmunoconjugate comprises the polypeptide sequences of SEQ ID NO: 205,SEQ ID NO: 241 and SEQ ID NO: 243, or variants thereof that retainfunctionality. In yet another embodiment, the immunoconjugate comprisesthe polypeptide sequences of SEQ ID NO: 205, SEQ ID NO: 241 and SEQ IDNO: 245, or variants thereof that retain functionality. In anotherspecific embodiment, the polypeptides are covalently linked, e.g., by adisulfide bond. In some embodiments the Fc domain subunits each comprisethe amino acid substitutions L234A, L235A, and P329G.

In a specific embodiment, the immunoconjugate comprises a polypeptidesequence encoded by a polynucleotide sequence that is at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequenceselected from the group of SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:200, SEQ ID NO: 204, SEQ ID NO: 210, SEQ ID NO: 244, SEQ ID NO: 246, SEQID NO: 270, SEQ ID NO: 272 and SEQ ID NO: 274. In another specificembodiment, the immunoconjugate comprises a polypeptide sequence encodedby a polynucleotide sequence selected from the group of SEQ ID NO: 196,SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 204, SEQ ID NO: 210, SEQ IDNO: 244, SEQ ID NO: 246, SEQ ID NO: 270, SEQ ID NO: 272 and SEQ ID NO:274. In another specific embodiment, the immunoconjugate comprises apolypeptide sequence encoded by a polynucleotide sequence that is atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to asequence selected from the group of SEQ ID NO: 194, SEQ ID NO: 202, SEQID NO: 208 and SEQ ID NO: 242. In yet another specific embodiment, theimmunoconjugate comprises a polypeptide sequence encoded by apolynucleotide sequence selected from the group of SEQ ID NO: 194, SEQID NO: 202, SEQ ID NO: 208 and SEQ ID NO: 242. In another specificembodiment, the immunoconjugate comprises a polypeptide sequence encodedby a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 206 or SEQID NO: 212. In yet another specific embodiment, the immunoconjugatecomprises a polypeptide sequence encoded by the polynucleotide sequenceof SEQ ID NO: 206 or SEQ ID NO: 212.

In one embodiment, the immunoconjugate comprises at least one, typicallytwo or more antigen binding moieties that are specific for theCarcinoembryonic Antigen (CEA). In a specific embodiment, the antigenbinding moieties of the immunoconjugate comprise a heavy chain variableregion sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% identical to the sequence of SEQ ID NO: 191 or SEQ IDNO: 295, or a variant thereof that retains functionality. In anotherspecific embodiment, the antigen binding moieties of the immunoconjugatecomprise a light chain variable region sequence that is at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequenceof SEQ ID NO: 189 or SEQ ID NO: 293, or a variant thereof that retainsfunctionality. In a more specific embodiment, the antigen bindingmoieties of the immunoconjugate comprise a heavy chain variable regionsequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to the sequence of SEQ ID NO: 191, or a variantthereof that retains functionality, and a light chain variable regionsequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to the sequence of SEQ ID NO: 189, or a variantthereof that retains functionality. In another specific embodiment, theantigen binding moieties of the immunoconjugate comprise a heavy chainvariable region sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 295, or avariant thereof that retains functionality, and a light chain variableregion sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% identical to the sequence of SEQ ID NO: 293, or avariant thereof that retains functionality.

In another specific embodiment, the heavy chain variable region sequenceof the antigen binding moieties of the immunoconjugate is encoded by apolynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to the sequence of SEQ ID NO: 192 or SEQ IDNO: 296. In yet another specific embodiment, the heavy chain variableregion sequence of the antigen binding moieties of the immunoconjugateis encoded by the polynucleotide sequence of SEQ ID NO: 192 or SEQ IDNO: 296. In another specific embodiment, the light chain variable regionsequence of the antigen binding moieties of the immunoconjugate isencoded by a polynucleotide sequence that is at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:190 or SEQ ID NO: 294. In yet another specific embodiment, the lightchain variable region sequence of the antigen binding moieties of theimmunoconjugate is encoded by the polynucleotide sequence of SEQ ID NO:190 or SEQ ID NO: 294.

In one embodiment, the immunoconjugate comprises a polypeptide sequencewherein a Fab heavy chain specific for CEA shares a carboxy-terminalpeptide bond with an Fc domain subunit comprising a knob modification,which in turn shares a carboxy-terminal peptide bond with an IL-2polypeptide. In a more specific embodiment, the immunoconjugatecomprises a polypeptide sequence selected from the group consisting ofSEQ ID NO: 229, SEQ ID NO: 275, SEQ ID NO: 277 and SEQ ID NO: 279, or avariant thereof that retains functionality. In one embodiment theimmunoconjugate comprises a polypeptide sequence wherein a Fab heavychain specific for CEA shares a carboxy-terminal peptide bond with an Fcdomain subunit comprising a hole modification. In a more specificembodiment, the immunoconjugate comprises the polypeptide sequence ofSEQ ID NO: 227 or SEQ ID NO: 281, or a variant thereof that retainsfunctionality. In another embodiment, the immunoconjugate comprises aFab light chain specific for CEA. In a more specific embodiment, theimmunoconjugate comprises the polypeptide sequence of SEQ ID NO: 231 orSEQ ID NO: 283, or a variant thereof that retains functionality. Inanother embodiment, the immunoconjugate comprises the polypeptidesequences of SEQ ID NO: 227, SEQ ID NO: 229 and SEQ ID NO: 231, orvariants thereof that retain functionality. In another embodiment, theimmunoconjugate comprises the polypeptide sequences of SEQ ID NO: 275,SEQ ID NO: 281 and SEQ ID NO: 283, or variants thereof that retainfunctionality. In another embodiment, the immunoconjugate comprises thepolypeptide sequences of SEQ ID NO: 277, SEQ ID NO: 281 and SEQ ID NO:283, or variants thereof that retain functionality. In anotherembodiment, the immunoconjugate comprises the polypeptide sequences ofSEQ ID NO: 279, SEQ ID NO: 281 and SEQ ID NO: 283, or variants thereofthat retain functionality. In another specific embodiment, thepolypeptides are covalently linked, e.g., by a disulfide bond. In someembodiments the Fc domain polypeptide chains comprise the amino acidsubstitutions L234A, L235A, and P329G.

In a specific embodiment, the immunoconjugate comprises a polypeptidesequence encoded by a polynucleotide sequence that is at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequenceselected from the group consisting of SEQ ID NO: 230, SEQ ID NO: 276,SEQ ID NO: 278 and SEQ ID NO: 280. In another specific embodiment, theimmunoconjugate comprises a polypeptide sequence encoded by thepolynucleotide sequence selected from the group consisting of SEQ ID NO:230, SEQ ID NO: 276, SEQ ID NO: 278 and SEQ ID NO: 280. In anotherspecific embodiment, the immunoconjugate comprises a polypeptidesequence encoded by a polynucleotide sequence that is at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence ofSEQ ID NO: 228 or SEQ ID NO: 282. In yet another specific embodiment,the immunoconjugate comprises a polypeptide sequence encoded by thepolynucleotide sequence of SEQ ID NO: 228 or SEQ ID NO: 282. In anotherspecific embodiment, the immunoconjugate comprises a polypeptidesequence encoded by a polynucleotide sequence that is at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence ofSEQ ID NO: 232 or SEQ ID NO: 284. In yet another specific embodiment,the immunoconjugate comprises a polypeptide sequence encoded by thepolynucleotide sequence of SEQ ID NO: 232 or SEQ ID NO: 284.

In some embodiments the immunoconjugate comprises a polypeptide sequencewherein an effector moiety polypeptide shares a carboxy-terminal peptidebond with an Fc domain subunit comprising a knob modification. In a morespecific embodiment, the immunoconjugate comprises a polypeptidesequence selected from the group of SEQ ID NO: 247, SEQ ID NO: 249 andSEQ ID NO: 251, or a variant thereof that retains functionality. In onesuch embodiment the immunoconjugate further comprises a polypeptidesequence wherein a Fab heavy chain specific for FAP shares acarboxy-terminal peptide bond with an Fc domain subunit comprising ahole modification. In a more specific embodiment, the immunoconjugatefurther comprises a polypeptide sequence selected from the group of SEQID NO: 193, SEQ ID NO: 201 and SEQ ID NO: 207, or a variant thereof thatretains functionality. In another such embodiment the immunoconjugatefurther comprises a polypeptide sequence wherein a Fab heavy chainspecific for EDB, TNC A1, TNC A2 or CEA shares a carboxy-terminalpeptide bond with an Fc domain subunit comprising a hole modification.In some embodiments the Fc domain subunits each comprise the amino acidsubstitutions L234A, L235A, and P329G. According to any of the aboveembodiments the immunoconjugate may further comprise a Fab light chainspecific for the corresponding antigen.

Immunoconjugates of the invention include those that have sequences thatare at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the sequences set forth in SEQ ID NOs 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67,69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185,187, 189, 191, 293, 295, 193, 195, 197, 199, 201, 203, 205, 207, 209,211, 213, 215, 217, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245,247, 249, 251, 269, 271, 273, 275, 277, 279, 281, 283, 285 and 287,including functional fragments or variants thereof. The invention alsoencompasses immunoconjugates comprising these sequences withconservative amino acid substitutions.

Polynucleotides

The invention further provides isolated polynucleotides encoding animmunoconjugate as described herein or a fragment thereof.

Polynucleotides of the invention include those that are at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequences set forth in SEQ ID NOs 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,294, 296, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216,218, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,270, 272, 274, 276, 278, 280, 282, 284, 286 and 288, includingfunctional fragments or variants thereof.

The polynucleotides encoding immunoconjugates of the invention may beexpressed as a single polynucleotide that encodes the entireimmunoconjugate or as multiple (e.g., two or more) polynucleotides thatare co-expressed. Polypeptides encoded by polynucleotides that areco-expressed may associate through, e.g., disulfide bonds or other meansto form a functional immunoconjugate. For example, the light chainportion of an antigen binding moiety may be encoded by a separatepolynucleotide from the portion of the immunoconjugate comprising theheavy chain portion of the antigen binding moiety, an Fc domain subunitand optionally the effector moiety. When co-expressed, the heavy chainpolypeptides will associate with the light chain polypeptides to formthe antigen binding moiety. In another example, the portion of theimmunoconjugate comprising the heavy chain portion of a first antigenbinding moiety, one of the two Fc domain subunits and the effectormoiety could be encoded by a separate polynucleotide from the portion ofthe immunoconjugate comprising the heavy chain portion of a secondantigen binding moiety and the other of the two Fc domain subunits. Whenco-expressed, the Fc domain subunits will associate to form the Fcdomain.

In one embodiment, an isolated polynucleotide of the invention encodes afragment of an immunoconjugate comprising a first antigen bindingmoiety, an Fc domain consisting of two subunits, and a single effectormoiety, wherein the antigen binding moiety is an antigen binding domaincomprising a heavy chain variable region and a light chain variableregion, particularly a Fab molecule. In one embodiment, an isolatedpolynucleotide of the invention encodes the heavy chain of the firstantigen binding moiety, a subunit of the Fc domain, and the effectormoiety. In another embodiment, an isolated polynucleotide of theinvention encodes the heavy chain of the first antigen binding moietyand a subunit of the Fc domain. In yet another embodiment, an isolatedpolynucleotide of the invention encodes a subunit of the Fc domain andthe effector moiety. In a more specific embodiment the isolatedpolynucleotide encodes a polypeptide wherein a Fab heavy chain shares acarboxy-terminal peptide bond with an Fc domain subunit.

In another specific embodiment the isolated polynucleotide encodes apolypeptide wherein an Fc domain subunit shares a carboxy-terminalpeptide bond with an effector moiety polypeptide. In yet anotherspecific embodiment, the isolated polynucleotide encodes a polypeptidewherein a Fab heavy chain shares a carboxy-terminal peptide bond with anFc domain subunit, which in turn shares a carboxy-terminal peptide bondwith an effector moiety polypeptide. In yet another specific embodimentthe isolated polynucleotide encodes a polypeptide wherein an effectormoiety polypeptide shares a carboxy-terminal peptide bond with an Fcdomain subunit.

In another embodiment, the present invention is directed to an isolatedpolynucleotide encoding an immunoconjugate or fragment thereof, whereinthe polynucleotide comprises a sequence that encodes a variable regionsequence as shown in SEQ ID NO 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165,167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 293 or295. In another embodiment, the present invention is directed to anisolated polynucleotide encoding an immunoconjugate or fragment thereof,wherein the polynucleotide comprises a sequence that encodes apolypeptide sequence as shown in SEQ ID NO 193, 195, 197, 199, 201, 203,205, 207, 209, 211, 213, 215, 217, 227, 229, 231, 233, 235, 237, 239,241, 243, 245, 247, 249, 251, 269, 271, 273, 275, 277, 279, 281, 283,285 or 287. In another embodiment, the invention is further directed toan isolated polynucleotide encoding an immunoconjugate or fragmentthereof, wherein the polynucleotide comprises a sequence that is atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to anucleotide sequence shown SEQ ID NO 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,294, 296, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216,218, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,270, 272, 274, 276, 278, 280, 282, 284, 286 or 288. In anotherembodiment, the invention is directed to an isolated polynucleotideencoding an immunoconjugate or fragment thereof, wherein thepolynucleotide comprises a nucleic acid sequence shown in SEQ ID NO 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,182, 184, 186, 188, 190, 192, 294, 296, 194, 196, 198, 200, 202, 204,206, 208, 210, 212, 214, 216, 218, 228, 230, 232, 234, 236, 238, 240,242, 244, 246, 248, 250, 252, 270, 272, 274, 276, 278, 280, 282, 284,286 or 288. In another embodiment, the invention is directed to anisolated polynucleotide encoding an immunoconjugate or fragment thereof,wherein the polynucleotide comprises a sequence that encodes a variableregion sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to an amino acid sequence of SEQ ID NO 23, 25, 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,53, 55, 57, 59, 61,63,65, 67, 69, 71,73, 75, 77, 79, 81,83, 85, 87, 89, 91, 93, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183,185, 187, 189, 191, 293 or 295. In another embodiment, the invention isdirected to an isolated polynucleotide encoding an immunoconjugate orfragment thereof, wherein the polynucleotide comprises a sequence thatencodes a polypeptide sequence that is at least 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO 193,195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 227, 229,231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 269, 271, 273,275, 277, 279, 281, 283, 285 or 287. The invention encompasses anisolated polynucleotide encoding an immunoconjugate or fragment thereof,wherein the polynucleotide comprises a sequence that encodes thevariable region sequences of SEQ ID NO 23, 25, 27, 29, 31, 33, 35, 37,39, 41, 43, 45, 47, 49, 51,53, 55, 57, 59, 61,63, 65, 67, 69, 71,73, 75,77, 79, 81,83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165,167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 293 or295 with conservative amino acid substitutions. The invention alsoencompasses an isolated polynucleotide encoding an immunoconjugate ofthe invention or fragment thereof, wherein the polynucleotide comprisesa sequence that encodes the polypeptide sequences of SEQ ID NO 193, 195,197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 227, 229, 231,233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 269, 271, 273, 275,277, 279, 281, 283, 285 or 287 with conservative amino acidsubstitutions.

In certain embodiments the polynucleotide or nucleic acid is DNA. Inother embodiments, a polynucleotide of the present invention is RNA, forexample, in the form of messenger RNA (mRNA). RNA of the presentinvention may be single stranded or double stranded.

Untargeted Conjugates

The invention provides not only immunoconjugates targeted to a specificantigen (e.g. a tumor antigen) but also untargeted conjugates comprisingone or more Fab molecules which do not specifically bind to any antigen,particularly not bind to any human antigen. The absence of specificbinding of these conjugates to any antigen (i.e. the absence of anybinding that can be discriminated from non-specific interaction) can bemeasured e.g. by ELISA or surface plasmon resonance as described herein.Such conjugates are particularly useful e.g. for enhancing the serumhalf life of the effector moiety they comprise, as compared to the serumhalf-life of the unconjugated effector moiety, where targeting to aparticular tissue is not desired.

Specifically, the invention provides a conjugate comprising a first Fabmolecule which does not specifically bind any antigen, an Fc domainconsisting of two subunits, and an effector moiety, wherein not morethan one effector moiety is present. More specifically, the inventionprovides a conjugate comprising a first Fab molecule comprising theheavy chain variable region sequence of SEQ ID NO: 299 and the lightchain variable region sequence of SEQ ID NO: 297, an Fc domainconsisting of two subunits, and an effector moiety, wherein not morethan one effector moiety is present. Like the immunoconjugates of theinvention, the conjugates can have a variety of configurations, asdescribed above under “Immunoconjugate Formats” (the antigen bindingmoiety of the immunoconjugate being replaced by a Fab molecule whichdoes not specifically bind to any antigen, such as a Fab moleculecomprising the heavy chain variable region sequence of SEQ ID NO: 299and the light chain variable region sequence of SEQ ID NO: 297).Likewise, the features of the Fc domain as well as the effector moietyas described above under “Fc domain” and “Effector moieties” for theimmunoconjugates of the invention equally apply, alone or incombination, to the untargeted conjugates of the invention.

In a particular embodiment, the conjugate comprises (i) animmunoglobulin molecule, comprising a first and a second Fab moleculewhich do not specifically bind any antigen and an Fc domain, and (ii) aneffector moiety, wherein not more than one effector moiety is presentand wherein the immunoglobulin molecule is a human IgG1 subclassimmunoglobulin; the Fc domain comprises a knob modification in one and ahole modification in the other one of its two subunits, and the aminoacid substitutions L234A, L235A and P329G in each of its subunits; andthe effector moiety is an IL-2 molecule fused to the carboxy-terminalamino acid of one of the immunoglobulin heavy chains, optionally througha linker peptide. In a specific embodiment, the conjugate comprises theheavy chain variable region sequence of SEQ ID NO: 299 and the lightchain variable region sequence of SEQ ID NO: 297.

In certain embodiments, the conjugate comprises (i) an immunoglobulinmolecule, comprising the heavy chain variable region sequence of SEQ IDNO: 299 and the light chain variable region sequence of SEQ ID NO: 297,and (ii) an effector moiety, wherein not more than one effector moietyis present. In one such embodiment the immunoglobulin molecule is ahuman IgG1 subclass immunoglobulin. In one such embodiment the Fc domaincomprises a knob modification in one and a hole modification in theother one of its two subunits. In a specific such embodiment, the Fcdomain comprises the amino acid substitutions L234A, L235A and P329G ineach of its subunits. In yet another such embodiment, the effectormoiety is an IL-2 molecule fused to the carboxy-terminal amino acid ofone of the immunoglobulin heavy chains, optionally through a linkerpeptide.

In one embodiment the conjugate comprises a polypeptide sequence whereina Fab heavy chain which does not specifically bind to any antigen sharesa carboxy-terminal peptide bond with an Fc domain subunit comprising aknob modification, which in turn shares a carboxy-terminal peptide bondwith an IL-2 polypeptide. In a more specific embodiment, the conjugatecomprises a polypeptide sequence selected from the group of SEQ ID NO:221, SEQ ID NO: 223, SEQ ID NO: 289 and SEQ ID NO: 291, or a variantthereof that retains functionality. In one embodiment the conjugatecomprises a polypeptide sequence wherein a Fab heavy chain which doesnot specifically bind to any antigen shares a carboxy-terminal peptidebond with an Fc domain subunit comprising a hole modification. In a morespecific embodiment, the conjugate comprises the polypeptide sequence ofSEQ ID NO: 219, or a variant thereof that retains functionality. Inanother embodiment, the conjugate comprises a Fab light chain which doesnot specifically bind any antigen. In a more specific embodiment, theconjugate comprises the polypeptide sequence of SEQ ID NO: 225, or avariant thereof that retains functionality. In another embodiment, theconjugate comprises the polypeptide sequences of SEQ ID NO: 219, SEQ IDNO: 221 and SEQ ID NO: 225, or variants thereof that retainfunctionality. In another embodiment, the conjugate comprises thepolypeptide sequences of SEQ ID NO: 219, SEQ ID NO: 223 and SEQ ID NO:225, or variants thereof that retain functionality. In anotherembodiment, the conjugate comprises the polypeptide sequences of SEQ IDNO: 219, SEQ ID NO: 289 and SEQ ID NO: 225, or variants thereof thatretain functionality. In another embodiment, the conjugate comprises thepolypeptide sequences of SEQ ID NO: 219, SEQ ID NO: 291 and SEQ ID NO:225, or variants thereof that retain functionality. In another specificembodiment, the polypeptides are covalently linked, e.g., by a disulfidebond. In some embodiments the Fc domain polypeptide chains comprise theamino acid substitutions L234A, L235A, and P329G.

In a specific embodiment, the conjugate comprises a polypeptide sequenceencoded by a polynucleotide sequence that is at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected fromthe group consisting of SEQ ID NO: 298, SEQ ID NO: 300, SEQ ID NO: 220,SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 290 and SEQID NO: 292. In another specific embodiment, the immunoconjugatecomprises a polypeptide sequence encoded by the polynucleotide sequenceselected from the group consisting of SEQ ID NO: 298, SEQ ID NO: 300,SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ IDNO: 290 and SEQ ID NO: 292.

The invention also provides an isolated polynucleotide encoding theconjugate of the invention of a fragment thereof. In a specificembodiment, the isolated polynucleotide comprises a sequence that is atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical tothe a sequence selected from the group of SEQ ID NO: 298, SEQ ID NO:300, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQID NO: 290 and SEQ ID NO: 292. The invention further provides anexpression vector comprising the isolated polynucleotide, and a hostcell comprising the isolated polynucleotide or the expression vector ofthe invention. In another aspect is provided a method of producing theconjugate of the invention, comprising the steps of a) culturing thehost cell of the invention under conditions suitable for the expressionof the conjugate and b) recovering the conjugate. The invention alsoencompasses a conjugate produced by the method of the invention. Thedisclosure provided herein in relating to methods of producing theimmunoconjugates of the invention (see e.g. under “Recombinant Methods”)can equally be applied to the conjugates of the invention.

The invention further provides a pharmaceutical composition comprisingthe conjugate of the invention and a pharmaceutically acceptablecarrier. The disclosure provided herein in relating to pharmaceuticalcompositions of the immunoconjugates of the invention (see e.g. under“Compositions, Formulations, and Routes of Administration”) can equallybe applied to the conjugates of the invention. Furthermore, theconjugate can be employed in the methods of use described herein for theimmunoconjugates of the invention. The disclosure provided herein inrelating to methods of using the immunoconjugates of the invention inthe treatment of disease (see e.g. under “Therapeutic Methods andCompositions”, “Other Agents and Treatments” and “Articles ofmanufacture”) can equally be applied to the conjugates of the invention.

Recombinant Methods

Immunoconjugates of the invention may be obtained, for example, bysolid-state peptide synthesis (e.g. Merrifield solid phase synthesis) orrecombinant production. For recombinant production one or morepolynucleotide encoding the immunoconjugate (fragment), e.g., asdescribed above, is isolated and inserted into one or more vectors forfurther cloning and/or expression in a host cell. Such polynucleotidemay be readily isolated and sequenced using conventional procedures. Inone embodiment a vector, preferably an expression vector, comprising oneor more of the polynucleotides of the invention is provided. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing the coding sequence of animmunoconjugate (fragment) along with appropriatetranscriptional/translational control signals. These methods include invitro recombinant DNA techniques, synthetic techniques and in vivorecombination/genetic recombination. See, for example, the techniquesdescribed in Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL,Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al., CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and WileyInterscience, N.Y (1989). The expression vector can be part of aplasmid, virus, or may be a nucleic acid fragment. The expression vectorincludes an expression cassette into which the polynucleotide encodingthe immunoconjugate (fragment) (i.e. the coding region) is cloned inoperable association with a promoter and/or other transcription ortranslation control elements. As used herein, a “coding region” is aportion of nucleic acid which consists of codons translated into aminoacids. Although a “stop codon” (TAG, TGA, or TAA) is not translated intoan amino acid, it may be considered to be part of a coding region, ifpresent, but any flanking sequences, for example promoters, ribosomebinding sites, transcriptional terminators, introns, 5′ and 3′untranslated regions, and the like, are not part of a coding region. Twoor more coding regions can be present in a single polynucleotideconstruct, e.g. on a single vector, or in separate polynucleotideconstructs, e.g. on separate (different) vectors. Furthermore, anyvector may contain a single coding region, or may comprise two or morecoding regions, e.g. a vector of the present invention may encode one ormore polypeptides, which are post- or co-translationally separated intothe final proteins via proteolytic cleavage. In addition, a vector,polynucleotide, or nucleic acid of the invention may encode heterologouscoding regions, either fused or unfused to a polynucleotide encoding theimmunoconjugate (fragment) of the invention, or variant or derivativethereof. Heterologous coding regions include without limitationspecialized elements or motifs, such as a secretory signal peptide or aheterologous functional domain. An operable association is when a codingregion for a gene product, e.g. a polypeptide, is associated with one ormore regulatory sequences in such a way as to place expression of thegene product under the influence or control of the regulatorysequence(s). Two DNA fragments (such as a polypeptide coding region anda promoter associated therewith) are “operably associated” if inductionof promoter function results in the transcription of mRNA encoding thedesired gene product and if the nature of the linkage between the twoDNA fragments does not interfere with the ability of the expressionregulatory sequences to direct the expression of the gene product orinterfere with the ability of the DNA template to be transcribed. Thus,a promoter region would be operably associated with a nucleic acidencoding a polypeptide if the promoter was capable of effectingtranscription of that nucleic acid. The promoter may be a cell-specificpromoter that directs substantial transcription of the DNA only inpredetermined cells. Other transcription control elements, besides apromoter, for example enhancers, operators, repressors, andtranscription termination signals, can be operably associated with thepolynucleotide to direct cell-specific transcription. Suitable promotersand other transcription control regions are disclosed herein. A varietyof transcription control regions are known to those skilled in the art.These include, without limitation, transcription control regions, whichfunction in vertebrate cells, such as, but not limited to, promoter andenhancer segments from cytomegaloviruses (e.g. the immediate earlypromoter, in conjunction with intron-A), simian virus 40 (e.g. the earlypromoter), and retroviruses (such as, e.g. Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit â-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas inducible promoters (e.g. promoters inducible tetracyclins).Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,and elements derived from viral systems (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence). Theexpression cassette may also include other features such as an origin ofreplication, and/or chromosome integration elements such as retrovirallong terminal repeats (LTRs), or adeno-associated viral (AAV) invertedterminal repeats (ITRs).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. For example, if secretionof the immunoconjugate is desired, DNA encoding a signal sequence may beplaced upstream of the nucleic acid encoding an immunoconjugates of theinvention or a fragment thereof. According to the signal hypothesis,proteins secreted by mammalian cells have a signal peptide or secretoryleader sequence which is cleaved from the mature protein once export ofthe growing protein chain across the rough endoplasmic reticulum hasbeen initiated. Those of ordinary skill in the art are aware thatpolypeptides secreted by vertebrate cells generally have a signalpeptide fused to the N-terminus of the polypeptide, which is cleavedfrom the translated polypeptide to produce a secreted or “mature” formof the polypeptide. In certain embodiments, the native signal peptide,e.g. an immunoglobulin heavy chain or light chain signal peptide isused, or a functional derivative of that sequence that retains theability to direct the secretion of the polypeptide that is operablyassociated with it. Alternatively, a heterologous mammalian signalpeptide, or a functional derivative thereof, may be used. For example,the wild-type leader sequence may be substituted with the leadersequence of human tissue plasminogen activator (TPA) or mouseβ-glucuronidase. Exemplary amino acid and corresponding polynucleotidesequences of secretory signal peptides are shown in SEQ ID NOs 8-16.

DNA encoding a short protein sequence that could be used to facilitatelater purification (e.g. a histidine tag) or assist in labeling theimmunoconjugate may be included within or at the ends of theimmunoconjugate (fragment) encoding polynucleotide.

In a further embodiment, a host cell comprising one or morepolynucleotides of the invention is provided. In certain embodiments ahost cell comprising one or more vectors of the invention is provided.The polynucleotides and vectors may incorporate any of the features,singly or in combination, described herein in relation topolynucleotides and vectors, respectively. In one such embodiment a hostcell comprises (e.g. has been transformed or transfected with) a vectorcomprising a polynucleotide that encodes (part of) an immunoconjugate ofthe invention. As used herein, the term “host cell” refers to any kindof cellular system which can be engineered to generate theimmunoconjugates of the invention or fragments thereof. Host cellssuitable for replicating and for supporting expression ofimmunoconjugates are well known in the art. Such cells may betransfected or transduced as appropriate with the particular expressionvector and large quantities of vector containing cells can be grown forseeding large scale fermenters to obtain sufficient quantities of theimmunoconjugate for clinical applications. Suitable host cells includeprokaryotic microorganisms, such as E. coli, or various eukaryoticcells, such as Chinese hamster ovary cells (CHO), insect cells, or thelike. For example, polypeptides may be produced in bacteria inparticular when glycosylation is not needed. After expression, thepolypeptide may be isolated from the bacterial cell paste in a solublefraction and can be further purified. In addition to prokaryotes,eukaryotic microbes such as filamentous fungi or yeast are suitablecloning or expression hosts for polypeptide-encoding vectors, includingfungi and yeast strains whose glycosylation pathways have been“humanized”, resulting in the production of a polypeptide with apartially or fully human glycosylation pattern. See Gerngross, NatBiotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215(2006). Suitable host cells for the expression of (glycosylated)polypeptides are also derived from multicellular organisms(invertebrates and vertebrates). Examples of invertebrate cells includeplant and insect cells. Numerous baculoviral strains have beenidentified which may be used in conjunction with insect cells,particularly for transfection of Spodoptera frugiperda cells. Plant cellcultures can also be utilized as hosts. See e.g. U.S. Pat. Nos.5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describingPLANTIBODIES™ technology for producing antibodies in transgenic plants).Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293Tcells as described, e.g., in Graham et al., J Gen Virol 36, 59 (1977)),baby hamster kidney cells (BHK), mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol Reprod 23, 243-251 (1980)), monkeykidney cells (CV1), African green monkey kidney cells (VERO-76), humancervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo ratliver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2), mouse mammary tumor cells (MMT 060562), TRI cells (as described,e.g., in Mather et al., Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC 5cells, and FS4 cells. Other useful mammalian host cell lines includeChinese hamster ovary (CHO) cells, including dhff CHO cells (Urlaub etal., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell linessuch as YO, NS0, P3X63 and Sp2/0. For a review of certain mammalian hostcell lines suitable for protein production, see, e.g., Yazaki and Wu,Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press,Totowa, N.J.), pp. 255-268 (2003). Host cells include cultured cells,e.g., mammalian cultured cells, yeast cells, insect cells, bacterialcells and plant cells, to name only a few, but also cells comprisedwithin a transgenic animal, transgenic plant or cultured plant or animaltissue. In one embodiment, the host cell is a eukaryotic cell,preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell,a human embryonic kidney (HEK) cell or a lymphoid cell (e.g., Y0, NS0,Sp20 cell). Standard technologies are known in the art to expressforeign genes in these systems. Cells expressing a polypeptidecomprising either the heavy or the light chain of an antigen bindingdomain such as an antibody, may be engineered so as to also express theother of the antibody chains such that the expressed product is anantibody that has both a heavy and a light chain.

In one embodiment, a method of producing an immunoconjugate according tothe invention is provided, wherein the method comprises culturing a hostcell comprising a polynucleotide encoding the immunoconjugate, asprovided herein, under conditions suitable for expression of theimmunoconjugate, and recovering the immunoconjugate from the host cell(or host cell culture medium).

The components of the immunoconjugate are genetically fused to eachother. Immunoconjugates can be designed such that its components arefused directly to each other or indirectly through a linker sequence.The composition and length of the linker may be determined in accordancewith methods well known in the art and may be tested for efficacy.Examples of linker sequences between the effector moiety and the Fcdomain are found in the sequences shown in SEQ ID NO 195, 197, 199, 203,209, 215, 229, 235, 237, 243, 245, 247, 249, 251, 269, 271, 273, 275,277, 279 and 285. Additional sequences may also be included toincorporate a cleavage site to separate the individual components of thefusion if desired, for example an endopeptidase recognition sequence.

In certain embodiments the one or more antigen binding moieties of theimmunoconjugate comprise at least an antibody variable region capable ofbinding an antigenic determinant. Variable regions can form part of andbe derived from naturally or non-naturally occurring antibodies andfragments thereof. Methods to produce polyclonal antibodies andmonoclonal antibodies are well known in the art (see e.g. Harlow andLane, “Antibodies, a laboratory manual”, Cold Spring Harbor Laboratory,1988). Non-naturally occurring antibodies can be constructed using solidphase-peptide synthesis, can be produced recombinantly (e.g. asdescribed in U.S. Pat. No. 4,186,567) or can be obtained, for example,by screening combinatorial libraries comprising variable heavy chainsand variable light chains (see e.g. U.S. Pat. No. 5,969,108 toMcCafferty). Antigen binding moieties and methods for producing the sameare also described in detail in PCT publication WO 2011/020783, theentire content of which is incorporated herein by reference.

Any animal species of antibody, antibody fragment, antigen bindingdomain or variable region can be used in the immunoconjugates of theinvention. Non-limiting antibodies, antibody fragments, antigen bindingdomains or variable regions useful in the present invention can be ofmurine, primate, or human origin. If the immunoconjugate is intended forhuman use, a chimeric form of antibody may be used wherein the constantregions of the antibody are from a human. A humanized or fully humanform of the antibody can also be prepared in accordance with methodswell known in the art (see e. g. U.S. Pat. No. 5,565,332 to Winter).Humanization may be achieved by various methods including, but notlimited to (a) grafting the non-human (e.g., donor antibody) CDRs ontohuman (e.g. recipient antibody) framework and constant regions with orwithout retention of critical framework residues (e.g. those that areimportant for retaining good antigen binding affinity or antibodyfunctions), (b) grafting only the non-human specificity-determiningregions (SDRs or a-CDRs; the residues critical for the antibody-antigeninteraction) onto human framework and constant regions, or (c)transplanting the entire non-human variable domains, but “cloaking” themwith a human-like section by replacement of surface residues. Humanizedantibodies and methods of making them are reviewed, e.g., in Almagro andFransson, Front Biosci 13, 1619-1633 (2008), and are further described,e.g., in Riechmann et al., Nature 332, 323-329 (1988); Queen et al.,Proc Natl Acad Sci USA 86, 10029-10033 (1989); U.S. Pat. Nos. 5,821,337,7,527,791, 6,982,321, and 7,087,409; Jones et al., Nature 321, 522-525(1986); Morrison et al., Proc Natl Acad Sci 81, 6851-6855 (1984);Morrison and Oi, Adv Immunol 44, 65-92 (1988); Verhoeyen et al., Science239, 1534-1536 (1988); Padlan, Molec Immun 31(3), 169-217 (1994);Kashmiri et al., Methods 36, 25-34 (2005) (describing SDR (a-CDR)grafting); Padlan, Mol Immunol 28, 489-498 (1991) (describing“resurfacing”); Dall'Acqua et al., Methods 36, 43-60 (2005) (describing“FR shuffling”); and Osbourn et al., Methods 36, 61-68 (2005) and Klimkaet al., Br J Cancer 83, 252-260 (2000) (describing the “guidedselection” approach to FR shuffling). Human antibodies and humanvariable regions can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr Opin Pharmacol 5, 368-74 (2001) and Lonberg, Curr OpinImmunol 20, 450-459 (2008). Human variable regions can form part of andbe derived from human monoclonal antibodies made by the hybridoma method(see e.g. Monoclonal Antibody Production Techniques and Applications,pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). Human antibodies andhuman variable regions may also be prepared by administering animmunogen to a transgenic animal that has been modified to produceintact human antibodies or intact antibodies with human variable regionsin response to antigenic challenge (see e.g. Lonberg, Nat Biotech 23,1117-1125 (2005). Human antibodies and human variable regions may alsobe generated by isolating Fv clone variable region sequences selectedfrom human-derived phage display libraries (see e.g., Hoogenboom et al.in Methods in Molecular Biology 178, 1-37 (O'Brien et al., ed., HumanPress, Totowa, N.J., 2001); and McCafferty et al., Nature 348, 552-554;Clackson et al., Nature 352, 624-628 (1991)). Phage typically displayantibody fragments, either as single-chain Fv (scFv) fragments or as Fabfragments. A detailed description of the preparation of antigen bindingmoieties for immunoconjugates by phage display can be found in theExamples appended to PCT publication WO 2011/020783.

In certain embodiments, the antigen binding moieties useful in thepresent invention are engineered to have enhanced binding affinityaccording to, for example, the methods disclosed in PCT publication WO2011/020783 (see Examples relating to affinity maturation) or U.S. Pat.Appl. Publ. No. 2004/0132066, the entire contents of which are herebyincorporated by reference. The ability of the immunoconjugate of theinvention to bind to a specific antigenic determinant can be measuredeither through an enzyme-linked immunosorbent assay (ELISA) or othertechniques familiar to one of skill in the art, e.g. surface plasmonresonance technique (analyzed on a BIACORE T100 system) (Liljeblad, etal., Glyco J 17, 323-329 (2000)), and traditional binding assays(Heeley, Endocr Res 28, 217-229 (2002)). Competition assays may be usedto identify an antibody, antibody fragment, antigen binding domain orvariable domain that competes with a reference antibody for binding to aparticular antigen, e.g. an antibody that competes with the L19 antibodyfor binding to the Extra Domain B of fibronectin (EDB). In certainembodiments, such a competing antibody binds to the same epitope (e.g. alinear or a conformational epitope) that is bound by the referenceantibody. Detailed exemplary methods for mapping an epitope to which anantibody binds are provided in Morris (1996) “Epitope MappingProtocols,” in Methods in Molecular Biology vol. 66 (Humana Press,Totowa, N.J.). In an exemplary competition assay, immobilized antigen(e.g. EDB) is incubated in a solution comprising a first labeledantibody that binds to the antigen (e.g. L19 antibody) and a secondunlabeled antibody that is being tested for its ability to compete withthe first antibody for binding to the antigen. The second antibody maybe present in a hybridoma supernatant. As a control, immobilized antigenis incubated in a solution comprising the first labeled antibody but notthe second unlabeled antibody. After incubation under conditionspermissive for binding of the first antibody to the antigen, excessunbound antibody is removed, and the amount of label associated withimmobilized antigen is measured. If the amount of label associated withimmobilized antigen is substantially reduced in the test sample relativeto the control sample, then that indicates that the second antibody iscompeting with the first antibody for binding to the antigen. See Harlowand Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.).

Immunoconjugates prepared as described herein may be purified byart-known techniques such as high performance liquid chromatography, ionexchange chromatography, gel electrophoresis, affinity chromatography,size exclusion chromatography, and the like. The actual conditions usedto purify a particular protein will depend, in part, on factors such asnet charge, hydrophobicity, hydrophilicity etc., and will be apparent tothose having skill in the art. For affinity chromatography purificationan antibody, ligand, receptor or antigen can be used to which theimmunoconjugate binds. For example, for affinity chromatographypurification of immunoconjugates of the invention, a matrix with proteinA or protein G may be used. Sequential Protein A or G affinitychromatography and size exclusion chromatography can be used to isolatean immunoconjugate essentially as described in the Examples. The purityof the immunoconjugate can be determined by any of a variety of wellknown analytical methods including gel electrophoresis, high pressureliquid chromatography, and the like. For example, the heavy chain fusionproteins expressed as described in the Examples were shown to be intactand properly assembled as demonstrated by reducing SDS-PAGE (see e.g.FIGS. 4A-D). Three bands were resolved at approximately Mr 25,000, Mr50,000 and Mr 60,000, corresponding to the predicted molecular weightsof the immunoglobulin light chain, heavy chain and heavy chain/effectormoiety fusion protein.

Assays

Immunoconjugates provided herein may be identified, screened for, orcharacterized for their physical/chemical properties and/or biologicalactivities by various assays known in the art.

Affinity Assays

The affinity of the immunoconjugate for an effector moiety receptor(e.g. IL-10R or various forms of IL-2R), an Fc receptor, or a targetantigen, can be determined in accordance with the methods set forth inthe Examples by surface plasmon resonance (SPR), using standardinstrumentation such as a BIAcore instrument (GE Healthcare), andreceptors or target proteins such as may be obtained by recombinantexpression. Alternatively, binding of immunoconjugates for differentreceptors or target antigens may be evaluated using cell linesexpressing the particular receptor or target antigen, for example byflow cytometry (FACS). A specific illustrative and exemplary embodimentfor measuring binding affinity is described in the following and in theExamples below.

According to one embodiment, K_(D) is measured by surface plasmonresonance using a BIACORE® T100 machine (GE Healthcare) at 25° C. withligand (e.g. effector moiety receptor, Fc receptor or target antigen)immobilized on CMS chips. Briefly, carboxymethylated dextran biosensorchips (CMS, GE Healthcare) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Recombinant ligand is diluted with 10 mM sodium acetate, pH 5.5, to0.5-30 μg/ml before injection at a flow rate of 10 μl/minute to achieveapproximately 100-5000 response units (RU) of coupled protein. Followingthe injection of the ligand, 1 M ethanolamine is injected to blockunreacted groups. For kinetics measurements, three- to five-fold serialdilutions of immunoconjugate (range between ˜0.01 nM to 300 nM) areinjected in HBS-EP+(GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA,0.05% Surfactant P20, pH 7.4) at 25° C. at a flow rate of approximately30-50 μl/min. Association rates (k_(on)) and dissociation rates (kw) arecalculated using a simple one-to-one Langmuir binding model (BIACORE®T100 Evaluation Software version 1.1.1) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (K_(D)) is calculated as the ratio k_(off)/k_(on). See, e.g.,Chen et al., J Mol Biol 293, 865-881 (1999).

Activity Assays

Biological activity of the immunoconjugates of the invention can bemeasured by various assays as described in the Examples. Biologicalactivities may for example include the induction of proliferation ofeffector moiety receptor-bearing cells, the induction of signaling ineffector moiety receptor-bearing cells, the induction of cytokinesecretion by effector moiety receptor-bearing cells, and the inductionof tumor regression and/or the improvement of survival.

Compositions, Formulations, and Routes of Administration

In a further aspect, the invention provides pharmaceutical compositionscomprising any of the immunoconjugates provided herein, e.g., for use inany of the below therapeutic methods. In one embodiment, apharmaceutical composition comprises any of the immunoconjugatesprovided herein and a pharmaceutically acceptable carrier. In anotherembodiment, a pharmaceutical composition comprises any of theimmunoconjugates provided herein and at least one additional therapeuticagent, e.g., as described below.

Further provided is a method of producing an immunoconjugate of theinvention in a form suitable for administration in vivo, the methodcomprising (a) obtaining an immunoconjugate according to the invention,and (b) formulating the immunoconjugate with at least onepharmaceutically acceptable carrier, whereby a preparation ofimmunoconjugate is formulated for administration in vivo.

Pharmaceutical compositions of the present invention comprise atherapeutically effective amount of one or more immunoconjugatedissolved or dispersed in a pharmaceutically acceptable carrier. Thephrases “pharmaceutical or pharmacologically acceptable” refers tomolecular entities and compositions that are generally non-toxic torecipients at the dosages and concentrations employed, i.e. do notproduce an adverse, allergic or other untoward reaction whenadministered to an animal, such as, for example, a human, asappropriate. The preparation of a pharmaceutical composition thatcontains at least one immunoconjugate and optionally an additionalactive ingredient will be known to those of skill in the art in light ofthe present disclosure, as exemplified by Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, incorporated herein byreference. Moreover, for animal (e.g., human) administration, it will beunderstood that preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiological Standards or corresponding authorities in other countries.Preferred compositions are lyophilized formulations or aqueoussolutions. As used herein, “pharmaceutically acceptable carrier”includes any and all solvents, buffers, dispersion media, coatings,surfactants, antioxidants, preservatives (e.g. antibacterial agents,antifungal agents), isotonic agents, absorption delaying agents, salts,preservatives, antioxidants, proteins, drugs, drug stabilizers,polymers, gels, binders, excipients, disintegration agents, lubricants,sweetening agents, flavoring agents, dyes, such like materials andcombinations thereof, as would be known to one of ordinary skill in theart (see, for example, Remington's Pharmaceutical Sciences, 18th Ed.Mack Printing Company, 1990, pp. 1289-1329, incorporated herein byreference). Except insofar as any conventional carrier is incompatiblewith the active ingredient, its use in the therapeutic or pharmaceuticalcompositions is contemplated.

The composition may comprise different types of carriers depending onwhether it is to be administered in solid, liquid or aerosol form, andwhether it need to be sterile for such routes of administration asinjection. Immunoconjugates of the present invention (and any additionaltherapeutic agent) can be administered intravenously, intradermally,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostatically, intrasplenically, intrarenally,intrapleurally, intratracheally, intranasally, intravitreally,intravaginally, intrarectally, intratumorally, intramuscularly,intraperitoneally, subcutaneously, subconjunctivally, intravesicularlly,mucosally, intrapericardially, intraumbilically, intraocularally,orally, topically, locally, by inhalation (e.g. aerosol inhalation),injection, infusion, continuous infusion, localized perfusion bathingtarget cells directly, via a catheter, via a lavage, in cremes, in lipidcompositions (e.g. liposomes), or by other method or any combination ofthe forgoing as would be known to one of ordinary skill in the art (see,for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack PrintingCompany, 1990, incorporated herein by reference). Parenteraladministration, in particular intravenous injection, is most commonlyused for administering polypeptide molecules such as theimmunoconjugates of the invention.

Parenteral compositions include those designed for administration byinjection, e.g. subcutaneous, intradermal, intralesional, intravenous,intraarterial intramuscular, intrathecal or intraperitoneal injection.For injection, the immunoconjugates of the invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hanks' solution, Ringer's solution, or physiological salinebuffer. The solution may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, theimmunoconjugates may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use. Sterileinjectable solutions are prepared by incorporating the immunoconjugatesof the invention in the required amount in the appropriate solvent withvarious of the other ingredients enumerated below, as required.Sterility may be readily accomplished, e.g., by filtration throughsterile filtration membranes. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The composition must be stable under theconditions of manufacture and storage, and preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Itwill be appreciated that endotoxin contamination should be keptminimally at a safe level, for example, less that 0.5 ng/mg protein.Suitable pharmaceutically acceptable carriers include, but are notlimited to: buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Aqueous injectionsuspensions may contain compounds which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, dextran,or the like. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl cleats or triglycerides, or liposomes.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(18th Ed. Mack Printing Company, 1990). Sustained-release preparationsmay be prepared. Suitable examples of sustained-release preparationsinclude semipermeable matrices of solid hydrophobic polymers containingthe polypeptide, which matrices are in the form of shaped articles, e.g.films, or microcapsules. In particular embodiments, prolonged absorptionof an injectable composition can be brought about by the use in thecompositions of agents delaying absorption, such as, for example,aluminum monostearate, gelatin or combinations thereof.

In addition to the compositions described previously, theimmunoconjugates may also be formulated as a depot preparation. Suchlong acting formulations may be administered by implantation (forexample subcutaneously or intramuscularly) or by intramuscularinjection. Thus, for example, the immunoconjugates may be formulatedwith suitable polymeric or hydrophobic materials (for example as anemulsion in an acceptable oil) or ion exchange resins, or as sparinglysoluble derivatives, for example, as a sparingly soluble salt.

Pharmaceutical compositions comprising the immunoconjugates of theinvention may be manufactured by means of conventional mixing,dissolving, emulsifying, encapsulating, entrapping or lyophilizingprocesses. Pharmaceutical compositions may be formulated in conventionalmanner using one or more physiologically acceptable carriers, diluents,excipients or auxiliaries which facilitate processing of the proteinsinto preparations that can be used pharmaceutically. Proper formulationis dependent upon the route of administration chosen.

The immunoconjugates may be formulated into a composition in a free acidor base, neutral or salt form. Pharmaceutically acceptable salts aresalts that substantially retain the biological activity of the free acidor base. These include the acid addition salts, e.g., those formed withthe free amino groups of a proteinaceous composition, or which areformed with inorganic acids such as for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric ormandelic acid. Salts formed with the free carboxyl groups can also bederived from inorganic bases such as for example, sodium, potassium,ammonium, calcium or ferric hydroxides; or such organic bases asisopropylamine, trimethylamine, histidine or procaine. Pharmaceuticalsalts tend to be more soluble in aqueous and other protic solvents thanare the corresponding free base forms.

Therapeutic Methods and Compositions

Any of the immunoconjugates provided herein may be used in therapeuticmethods. Immunoconjugates of the invention can be used asimmunotherapeutic agents, for example in the treatment of cancers.

For use in therapeutic methods, immunoconjugates of the invention wouldbe formulated, dosed, and administered in a fashion consistent with goodmedical practice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners.

In one aspect, immunoconjugates of the invention for use as a medicamentare provided. In further aspects, immunoconjugates of the invention foruse in treating a disease are provided. In certain embodiments,immunoconjugates of the invention for use in a method of treatment areprovided. In one embodiment, the invention provides an immunoconjugateas described herein for use in the treatment of a disease in anindividual in need thereof. In certain embodiments, the inventionprovides an immunoconjugate for use in a method of treating anindividual having a disease comprising administering to the individual atherapeutically effective amount of the immunoconjugate. In certainembodiments the disease to be treated is a proliferative disorder. In aparticular embodiment the disease is cancer. In other embodiments thedisease to be treated is an inflammatory disorder. In certainembodiments the method further comprises administering to the individuala therapeutically effective amount of at least one additionaltherapeutic agent, e.g., an anti-cancer agent if the disease to betreated is cancer. An “individual” according to any of the aboveembodiments is a mammal, preferably a human.

In a further aspect, the invention provides for the use of animmunconjugate of the invention in the manufacture or preparation of amedicament for the treatment of a disease in an individual in needthereof. In one embodiment, the medicament is for use in a method oftreating a disease comprising administering to an individual having thedisease a therapeutically effective amount of the medicament. In certainembodiments the disease to be treated is a proliferative disorder. In aparticular embodiment the disease is cancer. In other embodiments thedisease to be treated is an inflammatory disorder. In one embodiment,the method further comprises administering to the individual atherapeutically effective amount of at least one additional therapeuticagent, e.g., an anti-cancer agent if the disease to be treated iscancer. An “individual” according to any of the above embodiments may bea mammal, preferably a human.

In a further aspect, the invention provides a method for treating adisease in an individual, comprising administering to said individual atherapeutically effective amount of an immunoconjugate of the invention.In one embodiment a composition is administered to said individual,comprising immunoconjugate of the invention in a pharmaceuticallyacceptable form.

In certain embodiments the disease to be treated is a proliferativedisorder. In a particular embodiment the disease is cancer. In otherembodiments the disease to be treated is an inflammatory disorder. Incertain embodiments the method further comprises administering to theindividual a therapeutically effective amount of at least one additionaltherapeutic agent, e.g., an anti-cancer agent if the disease to betreated is cancer. An “individual” according to any of the aboveembodiments may be a mammal, preferably a human.

In certain embodiments the disease to be treated is a proliferativedisorder, particularly cancer. Non-limiting examples of cancers includebladder cancer, brain cancer, head and neck cancer, pancreatic cancer,lung cancer, breast cancer, ovarian cancer, uterine cancer, cervicalcancer, endometrial cancer, esophageal cancer, colon cancer, colorectalcancer, rectal cancer, gastric cancer, prostate cancer, blood cancer,skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer.Other cell proliferation disorders that can be treated using animmunoconjugate of the present invention include, but are not limited toneoplasms located in the: abdomen, bone, breast, digestive system,liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid,pituitary, testicles, ovary, thymus, thyroid), eye, head and neck,nervous system (central and peripheral), lymphatic system, pelvic, skin,soft tissue, spleen, thoracic region, and urogenital system. Alsoincluded are pre-cancerous conditions or lesions and cancer metastases.In certain embodiments the cancer is chosen from the group consisting ofrenal cell cancer, skin cancer, lung cancer, colorectal cancer, breastcancer, brain cancer, head and neck cancer. In some embodiments,particularly where the effector moiety of the immunoconjugate is IL-10,the disease to be treated is an inflammatory disorder. Non-limitingexamples of inflammatory disorders include rheumatoid arthritis,psoriasis or Crohn's disease. A skilled artisan readily recognizes thatin many cases the immunoconjugates may not provide a cure but may onlyprovide partial benefit. In some embodiments, a physiological changehaving some benefit is also considered therapeutically beneficial. Thus,in some embodiments, an amount of immunoconjugate that provides aphysiological change is considered an “effective amount” or a“therapeutically effective amount”. The subject, patient, or individualin need of treatment is typically a mammal, more specifically a human.

The immunoconjugates of the invention are also useful as diagnosticreagents. The binding of an immunoconjugate to an antigenic determinantcan be readily detected by using a secondary antibody specific for theeffector moiety. In one embodiment, the secondary antibody and theimmunoconjugate facilitate the detection of binding of theimmunoconjugate to an antigenic determinant located on a cell or tissuesurface.

In some embodiments, an effective amount of an immunoconjugate of theinvention is administered to a cell. In other embodiments, atherapeutically effective amount of an immunoconjugates of the inventionis administered to an individual for the treatment of disease. For theprevention or treatment of disease, the appropriate dosage of animmunoconjugate of the invention (when used alone or in combination withone or more other additional therapeutic agents) will depend on the typeof disease to be treated, the route of administration, the body weightof the patient, the type of immunoconjugate, the severity and course ofthe disease, whether the immunoconjugate is administered for preventiveor therapeutic purposes, previous or concurrent therapeuticinterventions, the patient's clinical history and response to theimmunoconjugate, and the discretion of the attending physician. Thepractitioner responsible for administration will, in any event,determine the concentration of active ingredient(s) in a composition andappropriate dose(s) for the individual subject. Various dosing schedulesincluding but not limited to single or multiple administrations overvarious time-points, bolus administration, and pulse infusion arecontemplated herein.

The immunoconjugate is suitably administered to the patient at one timeor over a series of treatments. Depending on the type and severity ofthe disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) ofimmunoconjugate can be an initial candidate dosage for administration tothe patient, whether, for example, by one or more separateadministrations, or by continuous infusion. One typical daily dosagemight range from about 1 μg/kg to 100 mg/kg or more, depending on thefactors mentioned above. For repeated administrations over several daysor longer, depending on the condition, the treatment would generally besustained until a desired suppression of disease symptoms occurs. Oneexemplary dosage of the immunoconjugate would be in the range from about0.005 mg/kg to about 10 mg/kg. In other non-limiting examples, a dosemay also comprise from about 1 microgram/kg body weight, about 5microgram/kg body weight, about 10 microgram/kg body weight, about 50microgram/kg body weight, about 100 microgram/kg body weight, about 200microgram/kg body weight, about 350 microgram/kg body weight, about 500microgram/kg body weight, about 1 milligram/kg body weight, about 5milligram/kg body weight, about 10 milligram/kg body weight, about 50milligram/kg body weight, about 100 milligram/kg body weight, about 200milligram/kg body weight, about 350 milligram/kg body weight, about 500milligram/kg body weight, to about 1000 mg/kg body weight or more peradministration, and any range derivable therein. In non-limitingexamples of a derivable range from the numbers listed herein, a range ofabout 5 mg/kg body weight to about 100 mg/kg body weight, about 5microgram/kg body weight to about 500 milligram/kg body weight, etc.,can be administered, based on the numbers described above. Thus, one ormore doses of about 0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg or 10 mg/kg (or anycombination thereof) may be administered to the patient. Such doses maybe administered intermittently, e.g. every week or every three weeks(e.g. such that the patient receives from about two to about twenty, ore.g. about six doses of the immunoconjugate). An initial higher loadingdose, followed by one or more lower doses may be administered. However,other dosage regimens may be useful. The progress of this therapy iseasily monitored by conventional techniques and assays.

The immunoconjugates of the invention will generally be used in anamount effective to achieve the intended purpose. For use to treat orprevent a disease condition, the immunoconjugates of the invention, orpharmaceutical compositions thereof, are administered or applied in atherapeutically effective amount. Determination of a therapeuticallyeffective amount is well within the capabilities of those skilled in theart, especially in light of the detailed disclosure provided herein.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays, such as cell culture assays. Adose can then be formulated in animal models to achieve a circulatingconcentration range that includes the IC₅₀ as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

Dosage amount and interval may be adjusted individually to provideplasma levels of the immunoconjugates which are sufficient to maintaintherapeutic effect. Usual patient dosages for administration byinjection range from about 0.1 to 50 mg/kg/day, typically from about 0.5to 1 mg/kg/day. Therapeutically effective plasma levels may be achievedby administering multiple doses each day. Levels in plasma may bemeasured, for example, by HPLC.

In cases of local administration or selective uptake, the effectivelocal concentration of the immunoconjugates may not be related to plasmaconcentration. One having skill in the art will be able to optimizetherapeutically effective local dosages without undue experimentation.

A therapeutically effective dose of the immunoconjugates describedherein will generally provide therapeutic benefit without causingsubstantial toxicity. Toxicity and therapeutic efficacy of animmunoconjugate can be determined by standard pharmaceutical proceduresin cell culture or experimental animals. Cell culture assays and animalstudies can be used to determine the LD₅₀ (the dose lethal to 50% of apopulation) and the ED₅₀ (the dose therapeutically effective in 50% of apopulation). The dose ratio between toxic and therapeutic effects is thetherapeutic index, which can be expressed as the ratio LD₅₀/ED₅₀.Immunoconjugates that exhibit large therapeutic indices are preferred.In one embodiment, the immunoconjugate according to the presentinvention exhibits a high therapeutic index. The data obtained from cellculture assays and animal studies can be used in formulating a range ofdosages suitable for use in humans. The dosage lies preferably within arange of circulating concentrations that include the ED₅₀ with little orno toxicity. The dosage may vary within this range depending upon avariety of factors, e.g., the dosage form employed, the route ofadministration utilized, the condition of the subject, and the like. Theexact formulation, route of administration and dosage can be chosen bythe individual physician in view of the patient's condition (see, e.g.,Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Ch.1, p. 1, incorporated herein by reference in its entirety).

The attending physician for patients treated with immunoconjugates ofthe invention would know how and when to terminate, interrupt, or adjustadministration due to toxicity, organ dysfunction, and the like.Conversely, the attending physician would also know to adjust treatmentto higher levels if the clinical response were not adequate (precludingtoxicity). The magnitude of an administered dose in the management ofthe disorder of interest will vary with the severity of the condition tobe treated, with the route of administration, and the like. The severityof the condition may, for example, be evaluated, in part, by standardprognostic evaluation methods. Further, the dose and perhaps dosefrequency will also vary according to the age, body weight, and responseof the individual patient.

Other Agents and Treatments

The immunoconjugates of the invention may be administered in combinationwith one or more other agents in therapy. For instance, animmunoconjugate of the invention may be co-administered with at leastone additional therapeutic agent. The term “therapeutic agent”encompasses any agent administered to treat a symptom or disease in anindividual in need of such treatment. Such additional therapeutic agentmay comprise any active ingredients suitable for the particularindication being treated, preferably those with complementary activitiesthat do not adversely affect each other. In certain embodiments, anadditional therapeutic agent is an immunomodulatory agent, a cytostaticagent, an inhibitor of cell adhesion, a cytotoxic agent, an activator ofcell apoptosis, or an agent that increases the sensitivity of cells toapoptotic inducers. In a particular embodiment, the additionaltherapeutic agent is an anti-cancer agent, for example a microtubuledisruptor, an antimetabolite, a topoisomerase inhibitor, a DNAintercalator, an alkylating agent, a hormonal therapy, a kinaseinhibitor, a receptor antagonist, an activator of tumor cell apoptosis,or an antiangiogenic agent.

Such other agents are suitably present in combination in amounts thatare effective for the purpose intended. The effective amount of suchother agents depends on the amount of immunoconjugate used, the type ofdisorder or treatment, and other factors discussed above. Theimmunoconjugates are generally used in the same dosages and withadministration routes as described herein, or about from 1 to 99% of thedosages described herein, or in any dosage and by any route that isempirically/clinically determined to be appropriate.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate compositions), and separate administration, in which case,administration of the immunoconjugate of the invention can occur priorto, simultaneously, and/or following, administration of the additionaltherapeutic agent and/or adjuvant. Immunoconjugates of the invention canalso be used in combination with radiation therapy.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an immunoconjugate of the invention. The label or packageinsert indicates that the composition is used for treating the conditionof choice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an immunoconjugate of the invention; and (b) a secondcontainer with a composition contained therein, wherein the compositioncomprises a further cytotoxic or otherwise therapeutic agent. Thearticle of manufacture in this embodiment of the invention may furthercomprise a package insert indicating that the compositions can be usedto treat a particular condition. Alternatively, or additionally, thearticle of manufacture may further comprise a second (or third)container comprising a pharmaceutically-acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1 General Methods Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook etal., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions. General information regarding the nucleotide sequences ofhuman immunoglobulins light and heavy chains is given in: Kabat, E. A.et al., (1991) Sequences of Proteins of Immunological Interest, FifthEd., NIH Publication No 91-3242.

DNA Sequencing

DNA sequences were determined by double strand sequencing.

Gene Synthesis

Desired gene segments where required were either generated by PCR usingappropriate templates or were synthesized by Geneart AG (Regensburg,Germany) from synthetic oligonucleotides and PCR products by automatedgene synthesis. In cases where no exact gene sequence was available,oligonucleotide primers were designed based on sequences from closesthomologues and the genes were isolated by RT-PCR from RNA originatingfrom the appropriate tissue. The gene segments flanked by singularrestriction endonuclease cleavage sites were cloned into standardcloning/sequencing vectors. The plasmid DNA was purified fromtransformed bacteria and concentration determined by UV spectroscopy.The DNA sequence of the subcloned gene fragments was confirmed by DNAsequencing. Gene segments were designed with suitable restriction sitesto allow sub-cloning into the respective expression vectors. Allconstructs were designed with a 5′-end DNA sequence coding for a leaderpeptide which targets proteins for secretion in eukaryotic cells. SEQ IDNOs 8-16 give exemplary leader peptides and polynucleotide sequencesencoding them.

Preparation of IL-2R βγ Subunit-Fc Fusions and IL-2R α Subunit Fc Fusion

To study IL-2 receptor binding affinity, a tool was generated thatallowed for the expression of a heterodimeric IL-2 receptor; theβ-subunit of the IL-2 receptor was fused to an Fc molecule that wasengineered to heterodimerize (Fc(hole)) (see SEQ ID NOs 17 and 18) usingthe “knobs-into-holes” technology (Merchant et al., Nat Biotech. 16,677-681 (1998)). The γ-subunit of the IL-2 receptor was then fused tothe Fc(knob) variant (see SEQ ID NOs 19 and 20), which heterodimerizedwith Fc(hole). This heterodimeric Fc-fusion protein was then used as asubstrate for analyzing the IL-2/IL-2 receptor interaction. The IL-2Rα-subunit was expressed as monomeric chain with an AcTev cleavage siteand an Avi His tag (SEQ ID NOs 21 and 22). The respective IL-2R subunitswere transiently expressed in HEK EBNA 293 with serum for the IL-2R βγsubunit construct and without serum for the α-subunit construct. TheIL-2R βγ subunit construct was purified on protein A (GE Healthcare),followed by size exclusion chromatography (GE Healthcare, Superdex 200).The IL-2R α-subunit was purified via His tag on a NiNTA column (Qiagen)followed by size exclusion chromatography (GE Healthcare, Superdex 75).Amino acid and corresponding nucleotide sequences of various receptorconstructs are given in SEQ ID NOs 17-22 and 255-268.

Preparation of Immunconjugates

Details about the generation and affinity maturation of antigen bindingmoieties directed to FAP can be found in the Examples appended to PCTpatent application publication no. WO 2012/020006, which is incorporatedherein by reference in its entirety. As described therein, variousantigen binding domains directed to FAP have been generated by phagedisplay, including the ones designated 4G8, 28H1 and 4B9 used in thefollowing examples. Clone 28H1 is an affinity matured antibody based onparental clone 4G8, while clone 4B9 is an affinity matured antibodybased on parental clone 3F2. The antigen binding domain designated 2B10used herein is directed to the A2 domain of Tenascin C (TNC A2). Detailsabout this and other antigen binding moieties directed against TNC A2can be found in PCT patent application publication no. WO 2012/020038,which is incorporated herein by reference in its entirety. The antigenbinding domain designated L19, directed against the Extra Domain B (EDB)of fibronectin is derived from the L19 antibody described in PCTpublication WO 2007/128563. The antigen binding domains designatedCH1A1A and CH1A1A 98/99 2F1 used herein are directed to CEA, and aredescribed in more detail in PCT patent application no.PCT/EP2012/053390, which is incorporated herein by reference in itsentirety.

The IL-2 quadruple mutant (qm) used as effector moiety in some of thefollowing examples is described in detail in PCT patent application no.PCT/EP2012/051991, which is incorporated herein by reference in itsentirety. Briefly, IL-2 qm is characterized by the following mutations:

-   -   1. T3A—knockout of predicted O-glycosylation site    -   2. F42A—knockout of IL-2/IL-2R α interaction    -   3. Y45A—knockout of IL-2/IL-2R α interaction    -   4. L72G—knockout of IL-2/IL-2R α interaction    -   5. C125A—mutation to avoid disulfide-bridged IL-2 dimers

The T3A mutation was chosen to eliminate the O-glycosylation site andobtain a protein product with higher homogeneity and purity when theIL-2 qm polypeptide or an immunoconjugate comprising it is expressed ineukaryotic cells such as CHO or HEK293 cells. The three mutations F42A,Y45A and L72G were chosen to interfere with the binding to CD25, theα-subunit of the IL-2 receptor. Reduced or abolished CD25 bindingresults in reduced activation-induced cell death (AICD), lack ofpreferential activation of regulatory T cells, as well as reducedtoxicity (as described in EP 11153964.9).

The DNA sequences were generated by gene synthesis and/or classicalmolecular biology techniques and subcloned into mammalian expressionvectors under the control of an MPSV promoter and upstream of asynthetic polyA site, each vector carrying an EBV OriP sequence.

Immunoconjugates as applied in the examples below were produced byco-transfecting exponentially growing HEK293-EBNA cells with themammalian expression vectors using calcium phosphate-transfection.Alternatively, HEK293 cells growing in suspension were transfected bypolyethylenimine (PEI) with the respective expression vectors.Alternatively, stably transfected CHO cell pools or CHO cell clones wereused for production in serum-free media. Subsequently, the IgG-cytokinefusion proteins were purified from the supernatant. Briefly,IgG-cytokine fusion proteins were purified by one affinity step withprotein A (HiTrap ProtA, GE Healthcare) equilibrated in 20 mM sodiumphosphate, 20 mM sodium citrate pH 7.5. After loading of thesupernatant, the column was first washed with 20 mM sodium phosphate, 20mM sodium citrate, pH 7.5 and subsequently washed with 13.3 mM sodiumphosphate, 20 mM sodium citrate, 500 mM sodium chloride, pH 5.45. TheIgG-cytokine fusion protein was eluted with 20 mM sodium citrate, 100 mMsodium chloride, 100 mM glycine, pH 3. Fractions were neutralized andpooled and purified by size exclusion chromatography (HiLoad 16/60Superdex 200, GE Healthcare) in final formulation buffer: 25 mMpotassium phosphate, 125 mM sodium chloride, 100 mM glycine pH 6.7.Exemplary detailed purification procedures and results are given forselected constructs below. The protein concentration of purified proteinsamples was determined by measuring the optical density (OD) at 280 nm,using the molar extinction coefficient calculated on the basis of theamino acid sequence. Purity and molecular weight of immunoconjugateswere analyzed by SDS-PAGE in the presence and absence of a reducingagent (5 mM 1,4-dithiotreitol) and stained with Coomassie blue(SimpleBlue™ SafeStain, Invitrogen). The NuPAGE® Pre-Cast gel system(Invitrogen) was used according to the manufacturer's instructions(4-20% Tris-glycine gels or 3-12% Bis-Tris). The aggregate content ofimmunoconjugate samples was analyzed using a Superdex 200 10/300GLanalytical size-exclusion column (GE Healthcare) in 2 mM MOPS, 150 mMNaCl, 0.02% NaN₃, pH 7.3 running buffer at 25° C. The integrity of theamino acid backbone of reduced antibody light and heavy chains can beverified by NanoElectrospray Q-TOF mass spectrometry after removal ofN-glycans by enzymatic treatment with Peptide-N Glycosidase F (RocheMolecular Biochemicals). The oligosaccharides attached to the Fc domainof the immunoconjugates are analysed by MALDI TOF-MS as described below.Oligosaccharides are enzymatically released from the immunoconjugates byPNGaseF digestion. The resulting digest solution containing the releasedoligosaccharides is either prepared directly for MALDI TOF-MS analysisor is further digested with EndoH glycosidase prior to samplepreparation for MALDI TOF-MS analysis.

Example 2

FAP-targeted IgG-IL-2 qm fusion proteins were generated based on theFAP-antibodies 4G8, 28H1 and 4B9, wherein one single IL-2 quadruplemutant (qm) was fused to the C-terminus of one heterodimeric heavy chainas shown in FIG. 2A. Targeting to the tumor stroma where FAP isselectively expressed is achieved via the bivalent antibody Fab region(avidity effect). Heterodimerization resulting in the presence of asingle IL-2 quadruple mutant is achieved by application of theknob-into-hole technology. In order to minimize the generation ofhomodimeric IgG-cytokine fusions the cytokine was fused to theC-terminus (with deletion of the C-terminal Lys residue) of theknob-containing IgG heavy chain via a (G₄S)₃ or G₄-(SG₄)₂ linker. Theantibody-cytokine fusion has IgG-like properties. To reduce FcγRbinding/effector function and prevent FcR co-activation, P329G L234AL235A (LALA) mutations were introduced in the Fc domain. The sequencesof these immunoconjugates are given SEQ ID NOs 193, 269 and 205 (28H1with (G₄S)₃ linker), SEQ ID NOs 193, 195 and 205 (28H1 with G₄-(SG₄)₂linker), SEQ ID NOs 201, 203 and 205 (4G8 with G₄-(SG₄)₂ linker), SEQ IDNOs 207, 209 and 211 (4B9 with G₄-(SG₄)₂ linker), SEQ ID NOs 207, 271and 211 (4B9 with (G₄S)₃ linker).

In addition, a CEA-targeted IgG-IL-2 qm fusion protein based on theanti-CEA antibody CH1A1A 98/99 2F1, a control DP47GS non-targetedIgG-IL-2 qm fusion protein wherein the IgG does not bind to a specifiedtarget, as well as a tumor stroma specific 2B10-based IgG-IL-2 qm fusionprotein targeted against the A2 domain of tenascin-C were generated. Thesequences of these immunoconjugates are given in SEQ ID NOs 275, 281 and283 (CH1A1A 98/99 2F1 with G₄-(SG₄)₂ linker), SEQ ID NOs 277, 281 and283 (CH1A1A 98/99 2F1 with (G₄S)₃ linker), SEQ ID NOs 219, 221 and 225(DP47GS with G₄-(SG₄)₂ linker), SEQ ID NOs 219, 289 and 225 (DP47GS with(G₄S)₃ linker), SEQ ID NOs 285, 287 and 239 (2B10 with (G₄S)₃ linker).The constructs were generated by transient expression in HEK293 EBNAcells and purified as described above. FIGS. 3A-D to FIGS. 9A-D showexemplary chromatograms and elution profiles of the purification (A, B)as well as the analytical SDS-PAGE and size exclusion chromatographiesof the final purified constructs (C, D). Transient expression yieldswere 42 mg/L for the 4G8-based, 20 mg/L for the 28H1-based, 10 mg/L forthe 4B9-based, 5.3 mg/L for the CH1A1A 98/99 2F1-based, 36.7 mg/L forthe 2B10-based and 13.8 mg/L for the DP47GS-based IgG-IL-2 qmimmunoconjugate.

In addition a 28H1-based FAP-targeted IgG-IL-15 immunoconjugate is beinggenerated, the sequences of which are given in SEQ ID NOs 193, 199 and205. In the IL-15 polypeptide sequence the glutamic acid residue atposition 53 is replaced by alanine to reduce binding to the α-subunit ofthe IL-15 receptor, and the asparagine residue at position 79 isreplaced by alanine to abolish glycosylation. The IgG-IL-15 fusionprotein is generated by transient expression and purified as describedabove.

FAP Binding Affinity

The FAP binding activity of the IgG-IL-2 qm immunoconjugates based on4G8 and 28H1 anti-FAP antibodies were determined by surface plasmonresonance (SPR) on a Biacore machine in comparison to the correspondingunmodified IgG antibodies. Briefly, an anti-His antibody (Penta-His,Qiagen 34660) was immobilized on CMS chips to capture 10 nM His-taggedhuman FAP (20 s). Temperature was 25° C. and HBS-EP was used as buffer.Analyte concentration was 50 nM down to 0.05 nM at a flow rate of 50μl/min (association: 300 s, dissociation: 900 s, regeneration: 60 s with10 mM glycine pH 2). Fitting was performed based on a 1:1 binding model,RI=0, Rmax=local (because of capture format). The following table givesthe estimated apperent bivalent affinities (pM avidity) as determined bySPR fitted with 1:1 binding RI=0, Rmax=local.

Hu FAP 4G8 IgG-IL-2 qm 100 pM 4G8 IgG  50 pM 28H1 IgG-IL-2 qm 175 pM28H1 IgG 200 pM

The data show that within the error of the method affinity for human FAPis retained for the 28H1-based immunoconjugate or only slightlydecreased for the 4G8-based immunoconjugate as compared to thecorresponding unmodified antibodies.

Similarly, the affinity (K_(D)) of 4B9 IgG-IL-2 qm (16 pM), CH1A1A 98/992F1 IgG-IL-2 qm (400 pM), CH1A1A 98/99 2F1 IgG-IL-2 wt (see Example 4;470 pM) and 2B10 IgG-IL-2 qm (150 pM, vs. 300 pM for unconjugated 2B10IgG) to human FAP, CEA and TNC A2, respectively, were determined by SPRat 25° C. Cross-reactivity of the 4B9 and 2B10 antibodies to human,murine and cynomolgus FAP or TNC A2, respectively, was also confirmed.

Subsequently, the affinity of the 4G8- and 28H1-based IgG-IL-2 qmimmunoconjugates to the IL-2R βγ heterodimer and the IL-2R α-subunitwere determined by surface plasmon resonance (SPR) in direct comparisonto the Fab-IL-2 qm-Fab immunoconjugate format described in PCT patentapplication no. PCT/EP2012/051991. Briefly, the ligands—either the humanIL-2R α-subunit or the human IL-2R βγ heterodimer—were immobilized on aCMS chip. Subsequently, the 4G8- and 28H1-based IgG-IL-2 qmimmunoconjugates or the 4G8- and 28H1-based Fab-IL-2 qm-Fabimmunoconjugates for comparison were applied to the chip as analytes at25° C. in HBS-EP buffer in concentrations ranging from 300 nM down to1.2 nM (1:3 dil.). Flow rate was 30 μl/min and the following conditionswere applied for association: 180 s, dissociation: 300 s, andregeneration: 2×30 s with 3 M MgCl₂ for IL-2R βγ heterodimer, 10 s with50 mM NaOH for IL-2R α-subunit. 1:1 binding was applied for fitting (1:1binding RI≠0, Rmax=local for IL-2R βγ, apparent K_(D), 1:1 binding RI=0,Rmax=local for IL-2R α). The respective K_(D) values are given in thetable below.

Apparent K_(D) [nM] Hu IL-2R βγ Hu IL-2R α 4G8 IgG-IL-2 qm 5.9 Nobinding 4G8 Fab-IL-2 qm-Fab 10.4 No binding 28H1 IgG-IL-2 qm 6.2 Nobinding 28H1 Fab-IL-2 qm-Fab 11.4 No binding

The data show that the 4G8- and 28H1-based IgG-IL-2 qm immunoconjugatesbind with at least as good affinity as the Fab-IL-2 qm-Fabimmunoconjugates to the IL-2R βγ heterodimer, whereas they do not bindto the IL-2R α-subunit due to the introduction of the mutationsinterfering with CD25 binding. Compared to the respective Fab-IL-2qm-Fab immunoconjugates the affinity of the IgG-IL-2 qm fusion proteinsappears to be slightly enhanced within the error of the method.

Similarly, the affinity of further constructs (4B9, DP47GS, 2B10, CH1A1A98/99 2F1) comprising either IL-2 wt (see Example 4) or IL-2 qm to theIL-2R βγ heterodimer and the IL-2R α-subunit was determined by SPR at25° C. For all constructs the apparent K_(D) for the human IL-2R βγheterodimer was between 6 and 12 nM (irrespective of whether theconstruct comprises IL-2 wt or IL-2 qm), whereas only the constructscomprising IL-2 wt bind to the IL-2R α-subunit at all (K_(D) for humanIL-2R α around 20 nM).

Biological Activity Assays with IgG-Cytokine Immunoconjugates

The biological activity of FAP-targeted 4G8-based IgG-IL-2 qm fusionswas investigated in several cellular assays in comparison tocommercially available IL-2 (Proleukin, Novartis/Chiron) and/or theFab-IL-2-Fab immunoconjugates described in EP 11153964.9.

Binding to FAP Expressing Cells

Binding of FAP-targeted 4G8-based IgG-IL-2 qm immunoconjugate to humanFAP expressed on stably transfected HEK293 cells was measured by FACS.Briefly, 250 000 cells per well were incubated with the indicatedconcentration of the immunoconjugate in a round-bottom 96-well plate,incubated for 30 min at 4° C., and washed once with PBS/0.1% BSA. Boundimmunoconjugate was detected after incubation for 30 min at 4° C. withFITC-conjugated AffiniPure F(ab′)2 Fragment goat anti-human F(ab′)2Specific (Jackson Immuno Research Lab #109-096-097, working solution:1:20 diluted in PBS/0.1% BSA, freshly prepared) using a FACS CantoII(Software FACS Diva). The results are shown in FIG. 10. The data showthat the IgG-IL-2 qm immunoconjugate binds to FAP-expressing cells withan EC50 value of 0.9 nM, comparable to that of the corresponding4G8-based Fab-IL-2 qm-Fab construct (0.7 nM).

IFN-γ Release by NK Cells (in Solution)

Subsequently, FAP-targeted 4G8-based IgG-IL-2 qm immunoconjugate wasstudied for the induction of IFN-γ release by NK92 cells as induced byactivation of IL-2R βγ signaling. Briefly, IL-2 starved NK92 cells (100000 cells/well in 96-U-well plate) were incubated with differentconcentrations of IL-2 immunoconjugate, comprising quadruple mutantIL-2, for 24 h in NK medium (MEM alpha from Invitrogen (#22561-021)supplemented with 10% FCS, 10% horse serum, 0.1 mM 2-mercaptoethanol,0.2 mM inositol and 0.02 mM folic acid). Supernatants were harvested andthe IFN-γ release was analysed using the anti-human IFN-γ ELISA Kit IIfrom Becton Dickinson (#550612). Proleukin (Novartis) and 28H1-basedFab-IL-2 qm-Fab served as positive control for IL-2-mediated activationof the cells. FIG. 11 shows that the FAP-targeted 4G8-based IgG-IL-2 qmimmunoconjugate is equally efficacious in inducing IFN-γ release as theaffinity matured 28H1-based Fab-IL-2 qm-Fab immunoconjugate.

STATS Phosphorylation Assay

In a last set of experiments we studied the effects of the FAP-targeted4G8-based IgG-IL-2 qm immunoconjugate on the induction of STATSphosphorylation compared to the 28H1 based Fab-IL-2-Fab and Fab-IL-2qm-Fab immunoconjugates as well as Proleukin on human NK cells, CD4⁺ Tcells, CD8⁺ T cells and T_(reg) cells from human PBMCs. Briefly, bloodfrom healthy volunteers was taken in heparin-containing syringes andPBMCs were isolated. PBMCs were treated with the indicatedimmunoconjuagtes at the indicated concentrations or with Proleukin(Novartis) as control. After 20 min incubation at 37° C., PBMCs werefixed with pre-warmed Cytofix buffer (Becton Dickinson #554655) for 10min at 37° C., followed by permeabilization with Phosflow Perm BufferIII (Becton Dickinson #558050) for 30 min at 4° C. Cells were washedtwice with PBS containing 0.1% BSA before FACS staining was performedusing mixtures of flow cytometry antibodies for detection of differentcell populations and phosphorylation of STATS. Samples were analysedusing a FACSCantoII with HTS from Becton Dickinson. NK cells weredefined as CD3⁻CD56⁺, CD8 positive T cells were defined as CD3⁺CD8⁺, CD4positive T cells were defined as CD4⁺CD25⁻CD127⁺ and T_(reg) cells weredefined as CD4⁺CD25⁺FoxP3⁺. For NK cells and CD8⁺ T cells that show noor very low CD25 expression (meaning that IL-2R signaling is mediatedprimarily via the IL-2R βγ heterodimer) the results show that the4G8-based IgG-IL-2 qm immunoconjugate was <10-fold less potent ininducing STATS phosphorylation than Proleukin, but slightly more potentthan 28H1-based Fab-IL-2-Fab and Fab-IL-2 qm-Fab immunoconjugates. OnCD4⁺ T cells, that show a rapid up-regulation of CD25 upon stimulation,the 4G8-based IgG-IL-2 qm immunoconjugate was less potent than the 28H1Fab-IL-2-Fab immunoconjugate, but slightly more potent than the 28H1Fab-IL-2 qm-Fab immunoconjugate, and still showed induction of IL-2Rsignaling at saturating concentrations comparable to Proleukin and 28H1Fab-IL-2-Fab. This is in contrast to T_(reg) cells where the potency ofthe 4G8-based IgG-IL-2 qm immunoconjugate was significantly reducedcompared to the Fab-IL-2-Fab immunoconjugate due to the high CD25expression on T_(reg) cells and the low binding affinity of the4G8-based IgG-IL-2 qm immunoconjugate to CD25. As a consequence of theabolishment of CD25 binding in the 4G8-based IgG-IL-2 qmimmunoconjugatee, IL-2 signaling in T_(reg) cells is only activated viathe IL-2R heterodimer at concentrations where IL-2R signaling isactivated on CD25-negative effector cells through the IL-2R βγheterodimer. Taken together the 4G8-based IgG-IL-2 qm immunoconjugatedescribed here is able to activate IL-2R signaling through the IL-2R βγheterodimer, but does not result in a preferential stimulation ofT_(reg) cells over other effector cells. The results of theseexperiments are shown in FIGS. 12A-D.

Binding of 2B10 IgG-IL-2 qm to TNC A2 Expressing Cells

Binding of TNC A2-targeted 2B10-based IgG-IL-2 qm immunoconjugate tohuman TNC A2 expressed on U87MG cells was measured by FACS. Briefly, 200000 cells per well were incubated with the indicated concentration ofthe immunoconjugate in a round-bottom 96-well plate, incubated for 30min at 4° C., and washed twice with PBS/0.1% BSA. Bound immunoconjugatewas detected after incubation for 30 min at 4° C. with FITC-conjugatedAffiniPure F(ab′)2 Fragment goat anti-human IgG Fcγ Specific (JacksonImmuno Research Lab #109-096-098, working solution: 1:20 diluted inPBS/0.1% BSA, freshly prepared) using a FACS CantoII (Software FACSDiva). The results are shown in FIG. 13. The data show that the 2B10IgG-IL-2 qm immunoconjugate binds to TNC A2-expressing U87MG cellsequally well as the corresponding unconjugated IgG.

Induction of NK92 Cell Proliferation by IgG-IL-2 Immunoconjugates

2B10 IgG-IL-2 qm, CH1A1A 98/99 2F1 IgG-IL-2 qm, CH1A1A 98/99 2F1IgG-IL-2 wt, 4B9 IgG-IL-2 qm and 4B9 IgG-IL-2 wt immunoconjugates weretested for their ability to induce proliferation of NK92 cells. Forproliferation assays, NK92 cells were starved in IL-2-free medium for 2hours, 10000 cells/well seeded into a flat-bottom 96-well plate and thenincubated for 3 days in a humidified incubator at 37° C., 5% CO₂ in thepresence of the IL-2 immunoconjugates ( ). After 3 days, the ATP contentof the cell lysates was measured using the CellTiter-Glo LuminescentCell Viability Assay from Promega (#G7571/2/3). The percentage of growthwas calculated setting a Proleukin (Novartis) concentration of 1.1 mg/mlto 100% proliferation and untreated cells without IL-2 stimulus to 0%proliferation. The results are shown in FIGS. 14 and 15. The data showthat all constructs were able to induce NK92 cell proliferation, withthe CH1A1A-based constructs being more active than the 2B10 IgG-IL-2 qmimmunoconjugate, and the constructs comprising IL-2 wt being more activethan the corresponding constructs with IL-2 qm.

Example 3

In general, the P329G LALA mutations that almost completely abolish FcγRinteraction of human IgG₁ antibodies (see European patent applicationno. EP 11160251.2, incorporated herein by reference in its entirety) areintroduced in order to reduce FcγR binding/effector function and thusprevent excessive cytokine release when the respective cytokinereceptors are co-activated with FcγR signaling. In specific cases, forexample when the antibody is targeting a highly tumor specific antigen,Fc effector functions may be retained by using an unmodified IgG Fcdomain or may be even further enhanced via glycoengineering of the IgGFc domain.

As an example thereof, we generated a CEA-targeted IgG-IL-2 qmimmunoconjugate where one single IL-2 quadruple mutant was fused to theC-terminus of one heterodimeric heavy chain via a (SG₄)₃-linker based onthe anti-CEA antibody clone CH1A1A. In this immunoconjugate the P329GLALA mutation was not included (see sequences of SEQ ID NOs 227, 229 and231). The immunoconjugate was expressed and purified as human wildtypeIgG- or glycoengineered IgG-IL-2 qm fusion protein as described below.

Preparation of (Glycoengineered) IgG-IL-2 qm Immunoconjugate

CEA-targeted CH1A1A-based IgG-IL-2 qm immunoconjugate was produced byco-transfecting HEK293-EBNA cells with the mammalian antibody expressionvectors. Exponentially growing HEK293-EBNA cells were transfected by thecalcium phosphate method. Alternatively, HEK293 cells growing insuspension are transfected by polyethylenimine. For the production ofunmodified non-glycoengineered IgG-IL-2 qm immunoconjugate, the cellswere transfected only with antibody heavy and light chain expressionvectors in a 1:1 ratio (wherein the antibody heavy chain vector is a 1:1mixture of two vectors: a vector for the heavy chain with the effectormoiety, and a vector for the heavy chain without effector moiety).

For the production of the glycoengineered CEA-targeted IgG-IL-2 qmimmunoconjugate, the cells were co-transfected with two additionalplasmids, one for expression of a GnTIII fusion polypeptide (a GnT-IIIexpression vector), and one for mannosidase II expression (a Golgimannosidase II expression vector) at a ratio of 4:4:1:1, respectively.Cells were grown as adherent monolayer cultures in T flasks using DMEMculture medium supplemented with 10% FCS, and were transfected when theyare between 50 and 80% confluent. For the transfection of a T150 flask,15 million cells were seeded 24 hours before transfection in 25 ml DMEMculture medium supplemented with FCS (at 10% v/v final), and cells wereplaced at 37° C. in an incubator with a 5% CO₂ atmosphere overnight. Foreach T150 flask to be transfected, a solution of DNA, CaCl₂ and waterwas prepared by mixing 94 μg total plasmid vector DNA divided equallybetween the light and heavy chain expression vectors, water to a finalvolume of 469 μl, and 469 μl of a 1M CaCl₂ solution. To this solution,938 μl of a 50 mM HEPES, 280 mM NaCl, 1.5 mM Na₂HPO₄ solution at pH 7.05were added, mixed immediately for 10 sec and left to stand at roomtemperature for 20 sec. The suspension was diluted with 10 ml of DMEMsupplemented with 2% FCS, and added to the T150 flask in place of theexisting medium. Then additional 13 ml of transfection medium wereadded. The cells were incubated at 37° C., 5% CO₂ for about 17 to 20hours, before the medium was replaced with 25 ml DMEM, 10% FCS. Theconditioned culture medium was harvested approximately 7 days after themedia exchange by centrifugation for 15 min at 210×g. The solution wassterile filtered (0.22 μm filter) and sodium azide in a finalconcentration of 0.01% w/v was added, and kept at 4° C.

The secreted wildtype or glycoengineered CEA IgG-IL-2 qmimmunoconjugates were purified from cell culture supernatants byaffinity chromatography using Protein A affinity chromatography,followed by a size exclusion chromatographic step on a HiLoad Superdex200 column (GE Healthcare) as described above. Protein concentration,purity, molecular weight, aggregate content and integrity were analysedas described above.

Oligosaccharide Structure Analysis of (Glycoengineered) IgG-IL-2 qmImmunoconjugates

For determination of the relative ratios of fucose-containing andnon-fucosylated oligosaccharide structures, released glycans of purifiedimmunoconjugate material are analyzed by MALDI TOF mass spectrometry.The immunoconjugate sample (about 50 μg) is incubated overnight at 37°C. with 5 mU N-glycosidase F (QAbio; PNGaseF: E-PNG01) in 2 mM Tris, pH7.0, in order to release the oligosaccharide from the protein backbone.For deamination of glycans acetic acid to a final concentration of 150mM is added and incubated for 1 h at 37° C. For analysis by MALDI TOFmass spectrometry, 2 μL of the sample are mixed on the MALDI target with2 μL DHB matrix solution (2, 5-dihydroxybenzoic acid [Bruker Daltonics#201346] dissolved in 50% ethanol/5 mM NaCl at 4 mg/ml) and analysedwith MALDI TOF Mass Spectrometer Autoflex II instrument (BrukerDaltonics). Routinely, 50-300 shots are recorded and summed up to asingle experiment. The spectra obtained are evaluated by the flexanalysis software (Bruker Daltonics) and masses are determined for theeach of the peaks detected. Subsequently, the peaks are assigned tofucose-containing or non-fucosylated carbohydrate structures bycomparing the masses calculated and the masses theoretically expectedfor the respective structures (e.g. complex, hybrid and oligo- orhigh-mannose, respectively, with and without fucose).

For determination of the ratio of hybrid structures, the antibodysamples are digested with N-glycosidase F and Endo-glycosidase H [QAbio;EndoH: E-EH02] concomitantly. N-glycosidase F releases all N-linkedglycan structures (complex, hybrid and oligo- and high mannosestructures) from the protein backbone and the Endo-glycosidase H cleavesall the hybrid type glycans additionally between the twoN-acetylglucosamine (GlcNAc) residues at the reducing end of the glycan.This digest is subsequently treated and analysed by MALDI TOF massspectrometry in the same way as described above for the N-glycosidase Fdigested sample. By comparing the pattern from the N-glycosidase Fdigest and the combined N-glycosidase F/Endo H digest, the degree ofreduction of the signals of a specific carbohydrate structure is used toestimate the relative content of hybrid structures. The relative amountof each carbohydrate structure is calculated from the ratio of the peakheight of an individual structure and the sum of the peak heights of alloligosaccharides detected. The amount of fucose is the percentage offucose-containing structures related to all carbohydrate structuresidentified in the N-glycosidase F treated sample (e.g. complex, hybridand oligo- and high-mannose structures). The degree of non-fucosylationis the percentage of structures lacking fucose relative to allcarbohydrates identified in the N-glycosidase F treated sample (e.g.complex, hybrid and oligo- and high-mannose structures).

Antibody-Dependent Cell-Mediated Cytotoxicity Assay

The wildtype and glycoengineered CEA-targeted CH1A1A IgG-IL-2 qmimmunoconjugates were compared in ADCC assays for their potential tomediate antibody mediated cellular cytotoxicity. Briefly,CEA-overexpressing A549 human tumor cells as target cells werecollected, washed and resuspended in culture medium, stained withfreshly prepared Calcein AM (Molecular Probes) at 37° C. for 30 min,washed three times, counted and diluted to 300 000 cells/ml. Thissuspension was transferred to a round-bottom 96-well plate (30000cells/well), the respective immunoconjugate dilution was added andincubated for 10 min to allow the binding of the tested immunoconjugateto the cells prior to contact with effector cells. Effector to targetratio was 25 to 1 for freshly isolated PBMCs. Co-incubation wasperformed for 4 hours. Two different read-out systems were used: therelease of lactate dehydrogenase (LDH) into supernatant afterdisintegration of the attacked cells, and the retention of Calcein inthe remaining living cells. LDH from co-culture supernatant wascollected and analyzed with a LDH detection Kit (Roche Applied Science).Substrate conversion by the LDH enzyme was measured with an ELISAabsorbance reader (SoftMaxPro software, reference wavelengths: 490 nmversus 650 nm). Residual Calcein in living cells was analyzed in afluorescence reader (Wallac VICTOR3 1420 Multilabel COUNTER (PerkinElmer)) after removing the rest of supernatant from pelletized cells,one washing step in PBS prior to lysis, and fixation of the cells byborate buffer (50 mM borate, 0.1% Triton).

FIG. 16 shows the result based on LDH detection. A similar result wasobtained based on the calcein retention (not shown). Both the constructswere able to mediate ADCC, the glycoengineered construct being similarlyactive as the corresponding glycoengineered unconjugated IgG. Asexpected, the non-glycoengineered construct showed reduced activity ascompared to the glycoengineered construct.

Example 4

FAP-targeted 28H1- or 4B9-based, CEA-targeted CH1A1A 98/99 2F1-based andnon-targeted DP47GS-based IgG-IL-2 immunoconjugates were generatedwherein one single wildtype IL-2 polypeptide is fused to the C-terminusof one heterodimeric heavy chain. Heterodimerization resulting in animmunoconjugate with a single IL-2 moiety was achieved by application ofthe knob-into-hole technology. In order to minimize the generation ofhomodimeric IgG-IL-2 fusions proteins the cytokine was fused to theknob-containing heavy chain (with deletion of the C-terminal Lysresidue) via a G₄-(SG₄)₂ or a (G₄S)₃ linker. The sequences of theseimmunoconjugates are given in SEQ ID NOs 193, 197 and 205 (28H1 withG₄-(SG₄)₂ linker) SEQ ID NOs 207, 273 and 211 (4B9 with (G₄S)₃ linker),SEQ ID NOs 277, 279 and 283 (CH1A1A 98/99 2F1 with (G₄S)₃ linker), SEQID NOs 219, 223 and 225 (DP47GS with G₄-(SG₄)₂ linker), SEQ ID NOs 219,293 and 225 (DP47GS with (G₄S)₃ linker). The antibody-cytokine fusionhas IgG-like properties. To reduce FcγR binding/effector function andprevent FcR co-activation, P329G LALA mutations were introduced in theFc domain. Both constructs were purified according to the methodsdescribed above. Final purification was done by size exclusionchromatography (HiLoad 26/60 Superdex 200, GE Healthcare) in the finalformulation buffer 20 mM histidine, 140 mM sodium chloride pH 6. FIGS.17A-D to FIGS. 20A-D show the respective chromatograms and elutionprofiles of the purification (A, B) as well as the analytical SDS-PAGEand size exclusion chromatographies of the final purified constructs (C,D). Yield was 15.6 mg/L for the untargeted DP47GS IgG-IL-2immunoconjugate, 26.7 mg/ml for the 28H1 IgG-IL-2 immunoconjugate, 4.6mg/L for the CH1A1A 98/99 2F1 IgG-IL-2 immunoconjugate and 11 mg/L forthe 4B9 IgG-IL-2 immunoconjugate.

Subsequently, their binding properties to FAP, respectively lack ofbinding, as well as binding to IL-2R βγ and IL-2R α chain weredetermined by SPR as described above (see Example 2). Cellular activityon immune effector cell populations and in vivo pharmacodynamic effectswere also studied.

Example 5

FAP-targeted 4G8-based as well as TNC A2-targeted 2B10-based IgG-IL-10immunoconjugates were constructed by fusing two different IL-10 cytokineformats to the C-terminus of the heavy chain of the heterodimeric IgGcomprising a hole modification: either a single-chain IL-10 wherein a(G₄S)₄ 20-mer linker was inserted between two IL-10 molecules, or anengineered monomeric IL-10 (Josephson et al., J Biol Chem 275, 13552-7(2000)). Both molecules were fused via a (G₄S)₃ 15-mer linker to theC-terminus of the heavy chain comprising a hole modification, withdeletion of the C-terminal Lys residue. Heterodimerization resulting inonly one heavy chain carrying an IL-10 moiety was achieved byapplication of the knob-into-hole technology. The IgG-cytokine fusionhas IgG-like properties. To reduce FcγR binding/effector function andprevent FcR co-activation, P329G LALA mutations were introduced in theFc domain of the immunoconjugate. The sequences of the respectiveconstructs are given in SEQ ID NOs 233, 235 and 239 (2B10 with scIL-10),SEQ ID NOs 233, 237 and 239 (2B10 with monomeric IL-10 “IL-10M1”), SEQID NOs 241, 243 and 205 (4G8 with scIL-10), SEQ ID NOs 241, 245 and 205(4G8 with IL-10M1). All these immunoconjugates were purified accordingto the methods described above. Subsequently, their binding propertiesto FAP or TNC A2, respectively, as well as their affinities to humanIL-10R1 were determined by SPR using the ProteOn XPR36 biosensor.Briefly, the targets FAP or TNC A2 as well as human IL-10R1 wereimmobilized in vertical orientation on the sensorchip surface (FAP bystandard amine coupling, TNC A2 and human IL-10R1 (both biotinylated viaa C-terminal avi-tag) by neutravidin-capture). Subsequently, theIgG-IL-10 immunoconjugates were injected in six differentconcentrations, including a zero-concentration, as analytes inhorizontal orientation.

After double-referencing, the sensorgrams were fit to a 1:1 interactionmodel to determine kinetic rate constants and affinities. The resultsfrom analytical SDS PAGE analysis and SPR-based affinity determinationsto target antigens as well as IL-10 receptor are shown in FIGS. 21A-Cand 22A-C. The data show that the immunoconjugates bind to TNC A2 or FAPwith K_(D) values of 52 or 26 pM, respectively, while K_(D) values forIL-10 receptor are 520 and 815 pM.

Example 6

According to the methods described above, IgG-cytokine fusion proteinswere generated and expressed consisting of one single 28H1-based or4B9-based Fab region directed to FAP fused to the N-terminus of an Fcdomain subunit comprising a hole modification, while the second Fabregion of the IgG heavy chain with the knob modification was replaced bya cytokine moiety via a (G₄S)_(n) linker (n=1). See FIG. 2C for aschematic representation of this immunoconjugate format (also referredto as “1+1” format). Cytokine moieties used were the IL-2 quadruplemutant described above and in PCT patent application no.PCT/EP2012/051991 (see SEQ ID NO: 3), IL-7 and IFN-α. Correspondingsequences of the fusion polypeptides comprising the cytokine moiety,fused to the N-terminus of an Fc domain subunit comprising a knobmodification via a linker peptide, are given in SEQ ID NOs 247(comprising quadruple mutant IL-2), 249 (comprising IL-7), and 251(comprising IFN-α). In these constructs, targeting of theimmunoconjugate is achieved via the high affinity monovalent Fab region.This format may be recommended in cases where internalization of theantigen may be reduced using a monovalent binder. The immunoconjugateswere produced, purified and analysed as described above. For constructscomprising IL-2 qm or IL-7, protein A affinity chromatography and sizeexclusion chromatography were combined in a single run. 20 mM histidine,140 mM NaCl pH 6.0 was used as size exclusion chromatography and finalformulation buffer. FIGS. 23A-C to 26A-D show the elution profiles andchromatograms of the purifications as well as the analytical SDS-PAGEand size exclusion chromatograms of the final purified constructs. Theyields were 11 mg/L for the 4B9 “1+1” IgG-IL-2 qm, 43 mg/L for the 28H1“1+1” IgG-IL-2 qm, 20.5 mg/L for the 4B9 “1+1” IgG-IL-7 and 10.5 mg/Lfor the 4B9 “1+1” IgG-IFN-α constructs.

The ability of “1+1” constructs comprising IL-2 qm to induce NK cellproliferation, compared to IgG-IL-2 qm immunoconjugates, was tested.NK-92 cells were starved for 2 h before seeding 10000 cells/well into96-well-black-flat-clear bottom plates. The immunoconjugates weretitrated onto the seeded NK-92 cells. After 72 h the ATP content wasmeasured to determine the number of viable cells using the“CellTiter-Glo Luminescent Cell Viability Assay” Kit (Promega) accordingto the manufacturer's instructions. FIG. 27 shows that the “1+1”constructs are able to induce proliferation of NK-92 cells, beingslightly less active than the corresponding IgG-IL-2 qm constructs.

The 4B9-based “1+1” constructs comprising IL-2 qm or IL-7 were testedfor their ability to induce T cell proliferation, compared to IgG-IL-2immunoconjugates. Peripheral blood mononuclear cells (PBMC) wereprepared using Histopaque-1077 (Sigma Diagnostics Inc., St. Louis, Mo.,USA). In brief, blood from buffy coats was diluted 5:1 with calcium- andmagnesium-free PBS, and layered on Histopaque-1077. The gradient wascentrifuged at 450×g for 30 min at room temperature (RT) without breaks.The interphase containing the PBMCs was collected and washed three timeswith PBS (350×g followed by 300×g for 10 min at RT). PBMCs werepre-stimulated with 1 μg/ml PHA-M (Sigma Aldrich #L8902) overnight,before they were labeled with 100 nM CFSE (carboxyfluoresceinsuccinimidyl ester) for 15 min at 37° C. Cells were washed with 20 mlmedium before recovering the labeled PBMCs for 30 min at 37° C. Thecells were washed, counted, and 100000 cells were seeded into96-well-U-bottom plates. The immunoconjugates were titrated onto theseeded cells for an incubation time of 6 days. Thereafter, cells werewashed, stained for appropriate cell surface markers, and analyzed byFACS using a BD FACSCantoII. CD4 T cells were defined as CD3⁺/CD8⁻, andCD8 T cells as CD3⁺/CD8⁺.

FIGS. 28A-B show that the “1+1” constructs comprising either IL-2 qm orIL-7 are able to induce proliferation of PHA-activated CD4 (FIG. 28A)and CD8 T cells (FIG. 28B). As for NK cells, the “1+1” constructcomprising IL-2 qm is slightly less active than an IgG-IL-2 qmconstruct.

The 4B9-based “1+1” construct comprising IFN-α was tested for itsability to inhibit Daudi cell proliferation, in comparison to Roferon A(Roche). Briefly, Daudi cells were labeled with 100 nM CFSE and seededinto a 96-well U-bottom plate (50′000 cells/well). The molecules wereadded at the indicated concentrations, followed by incubation for 3 daysat 37° C. Proliferation was measured by analyzing the CFSE dilution,excluding dead cells from analysis by use of life/dead stain.

FIG. 29 shows that the construct was able to inhibit proliferation ofDaudi cells, at least as potently as Roferon A.

Example 7

A single dose pharmacokinetics (PK) study was performed in tumor-freeimmunocompetent 129 mice for FAP-targeted IgG-IL2 immunoconjugatescomprising either wild type or quadruple mutant IL-2, and untargetedIgG-IL-2 immunoconjugates comprising either wild type or quadruplemutant IL-2.

Female 129 mice (Harlan, United Kingdom), aged 8-9 weeks at the start ofthe experiment, were maintained under specific-pathogen-free conditionswith daily cycles of 12 h light/12 h darkness according to committedguidelines (GV-Solas; Felasa; TierschG). The experimental study protocolwas reviewed and approved by local government (P 2008016). Afterarrival, animals were maintained for one week to get accustomed to thenew environment and for observation. Continuous health monitoring wascarried out on a regular basis.

Mice were injected i.v. once with FAP-targeted 28H1 IgG-IL2 wt (2.5mg/kg) or 28H1 IgG-IL2 qm (5 mg/kg), or untargeted DP47GS IgG-IL2 wt (5mg/kg) or DP47GS IgG-IL2 qm (5 mg/kg). All mice were injected i.v. with200 μl of the appropriate solution. To obtain the proper amount ofimmunoconjugate per 200 the stock solutions were diluted with PBS asnecessary.

Mice were bled at 1, 8, 24, 48, 72, 96 h; and every 2 days thereafterfor 3 weeks. Sera were extracted and stored at −20° C. until ELISAanalysis. Immunoconjugate concentrations in serum were determined usingan ELISA for quantification of the IL-2-immunoconjugate antibody(Roche-Penzberg). Absorption was measured using a measuring wavelengthof 405 nm and a reference wavelength of 492 nm (VersaMax tunablemicroplate reader, Molecular Devices).

FIGS. 30A-B show the pharmacokinetics of these IL-2 immunoconjugates.Both the FAP-targeted (FIG. 30A) and untargeted (FIG. 30B) IgG-IL2 qmconstructs have a longer serum half-life (approx. 30 h) than thecorresponding IgG-IL-2 wt constructs (approx. 15 h). Of note, althoughthe experimental conditions are not directly comparable, the serumhalf-life of the IL-2 immunoconjugates of the invention appears to belonger than the serum half-life of art-known “2+2” IgG-IL-2immunoconjugates (see FIG. 1) as reported e.g. in Gillies et al., ClinCancer Res 8, 210-216 (2002).

Concentration Compound Dose Formulation buffer (mg/mL) 28H1-IgG- 2.5mg/kg 20 mM Histidine, 3.84 IL2 wt 140 mM NaCl, (= stock solution) pH6.0 28H1-IgG-   5 mg/kg 20 mM Histidine, 2.42 IL2 qm    140 mM NaCl, (=stock solution)    pH 6.0 DP47GS-   5 mg/kg 20 mM Histidine, 3.74IgG-IL2wt    140 mM NaCl, (= stock solution)    pH 6.0 DP47GS-   5 mg/kg20 mM Histidine, 5.87 IgG- 140 mM NaCl, (= stock solution) IL2QM pH 6.0

Example 8

A biodistribution study was performed to assess tumor targeting of theimmunoconjugates of the invention. FAP-targeted 28H1-based IgG-IL-2 qmwas compared to FAP-targeted unconjugated 28H1 IgG and 4B9 IgG, anduntargeted DP47GS IgG. Furthermore, a SPECT/CT imaging study wasperformed with 4B9 IgG-IL-2 qm, compared to DP47GS IgG-IL-2 qm, 4B9 IgGand DP47GS IgG.

DTPA Conjugation and ¹¹¹In Labeling

Solutions of 28H1 IgG-IL-2 qm, 28H1 IgG₁, 4B9 IgG-IL-2 qm, 4B9 IgG₁ andDP47 IgG₁ were dialysed against phosphate buffered saline (PBS, 15 mM).Two mg of the constructs (5 mg/ml) were conjugated withisothiocyanatobenzyl-diethylenetriaminepentaacetic acid (ITC-DTPA,Macrocyclis, Dallas, Tex.) in 0.1 M NaHCO₃, pH 8.2, under strictmetal-free conditions, by incubation with a 5-fold molar excess ofITC-DTPA for one hour at room temperature (RT). Unconjugated ITC-DTPAwas removed by dialysis against 0.1 M 2-(N-morpholino)ethanesulfonicacid (MES) buffer, pH 5.5.

The purified conjugates were radiolabeled by incubation with ¹¹¹In(Covidien BV, Petten, The Netherlands) in 0.1 M MES buffer, pH 5.5containing 0.05% bovine serum albumin (BSA) and 0.05% Tween-80, at RT,under strict metal-free conditions for 30 min. After radiolabelingethylenediaminetetraacetic acid (EDTA) was added to a finalconcentration of 5 mM to chelate the unbound ¹¹¹In. The ¹¹¹In labeledproducts were purified by gelfiltration on disposable G25M columns(PD10, Amersham Biosciences, Uppsala, Sweden). Radiochemical purity ofpurified ¹¹¹In labeled constructs were determined by instant thin-layerchromatography (ITLC) on TEC Control chromatography strips (Biodex,Shirley, N.Y.), using 0.1 M citrate buffer, pH 6.0, as the mobile phase.The specific activity of the ¹¹¹In-labeled preparations was 0.6-4.6MBq/μg.

Lindmo Assay

The immunoreactive fraction of ¹¹¹In labeled antibody preparations wasdetermined as described previously (Lindmo et al. (1984) J ImmunolMethods 72, 77-89). Briefly, a serial dilution series of human embryonickidney (HEK) cells transfected with fibroblast activation protein (FAP)cDNA (HEK-FAP cells) were incubated with 200 Bq of the ¹¹¹In-labeledconstruct at 37° C. for 1 hour. A duplicate of the lowest cellconcentration was incubated in the presence of an excess of non-labeledconstruct to correct for non-specific binding. After incubation, thecells were washed, spun down and cell associated radioactivity wasdetermined in the cell pellet in a gamma-counter (Wallac Wizzard 3″ 1480automatic γ-counter, Pharmacia LKB). The immunoreactive fraction of thepreparations ranged between 75-94%.

Animals

Female BALB/c nude mice (8-9 weeks, +/−20 g) were purchased from Janvierand housed in the Central Animal Facility of the Radboud UniversityNijmegen Medical Centre under standard conditions with 5 animals inindividually ventilated cages with ad lib. access to food and water.After one week acclimatization the animals were inoculated s.c. with10×10⁶ HEK-FAP cells in matrigel (1:3) in the left flank and optionallywith 5×10⁶ HEK-293 cells in matrigel (1:3) in the right flank. Xenograftgrowth was monitored by caliper measurement(volume=(4/3·π)·(½·length)·(½·width)·(½·height). When xenografts reacheda volume of 100 mm³, mice were injected i.v. with the ¹¹¹In-labeledconstructs.

Biodistribution (28H1 IgG-IL-2 qm, 28H1 IgG₁, 4B9 IgG₁ and DP47GS IgG₁)

¹¹¹In-labeled constructs (5 MBq, 150 μg, 200 μl) were injected i.v. viathe tail vein. Twenty-four hours after injection the animals wereeuthanized by suffocation in CO₂/O₂ atmosphere. Blood, muscle,xenograft, lung, spleen, pancreas, kidney, stomach (empty), duodenum(empty) and liver were collected, weighed and radioactivity wasdetermined in a gamma-counter (Wallac Wizard). Standards of the injecteddose (1%) were counted simultaneously and tissue uptake was calculatedas % of the injected dose per gram tissue (% ID/g).

SPECT-CT Analysis (4B9 IgG-IL-2 qm, 4B9 IgG₁, DP47GS IgG-IL-2 qm andDP47GS IgG₁)

¹¹¹In-labeled 4B9-IgG-IL-2 qm, 4B9-IgG₁, DP47GS-IgG-IL-2 qm andDP47GS-IgG, were injected i.v. (20 MBq, 50, 150, 300 μg, 200 μl). At 4,24, 72 and 144 hours after injection the animals were anesthetized withisoflurane/O₂ and scanned for 30 to 60 min in a U-SPECT II microSPECT/CTcamera (MILabs, Utrecht, The Netherlands) equipped with a 1.0 mm mousecollimator. Computed tomography (CT) was performed directly after SPECT.Both SPECT (voxel size of 0.4 mm) and CT scans were reconstructed withMILabs software and SPECT and CT scans were co-registered to determineexact location of radio-signal. 3D images were created using SiemensInveon Research Workplace software.

FIG. 31 shows that there is no significant difference between tissuedistribution and tumor targeting of 28H1 and 4B9 IgG1 and 28H1 IgG-IL-2qm at 24 hours (hence the cytokine does not significantly alter thetissue distribution and tumor targeting properties of theimmunoconjugates), and that tumor-to-blood ratios for the FAP-targetedconstructs are significantly greater than for the non-targeted DP47GScontrol IgG.

These results were confirmed in SPECT/CT imaging for the 4B9 IgG-IL-2 qmimmunconjugate (data not shown). 4B9 IgG-IL-2 qm localized in theFAP-positive HEK-FAP but not in the FAP-negative HEK-293 control tumors,while the untargeted DP47GS immunoconjugate did not localize in eithertumor. Unlike with the unconjugated IgGs, a weak uptake of 4B9 IgG-IL-2qm was observed also in the spleen.

Example 9

Binding of 28H1-based IgG-IL-2 qm and a 28H1-based IgG-(IL-2 qm)₂ (i.e.a “2+2” format immunoconjugate as depicted in FIG. 1; sequences areshown in SEQ ID NOs 253 and 205) to NK 92 cells was compared. 200000NK92 cells per well were seeded in a 96-well plate. The immunoconjugateswere titrated onto the NK92 cells and incubated for 30 min at 4° C. toallow binding. The cells were washed twice with PBS containing 0.1% BSAto remove unbound constructs. For detection of the immunoconjugates aFITC-labeled anti-human Fc-specific antibody was added for 30 min at 4°C. The cells were again washed twice with PBS containing 0.1% BSA andanalyzed by FACS using a BD FACSCantoII.

As illustrated in FIG. 32, the “2+2” immunoconjugate shows betterbinding to NK 92 cells than the corresponding “2+1” construct.

Example 10 Induction of Human PBMC Proliferation by IL-2Immunoconjugates

Peripheral blood mononuclear cells (PBMC) were prepared usingHistopaque-1077 (Sigma Diagnostics Inc., St. Louis, Mo., USA). In brief,venous blood from healthy volunteers was drawn into heparinizedsyringes. The blood was diluted 2:1 with calcium- and magnesium-freePBS, and layered on Histopaque-1077. The gradient was centrifuged at450×g for 30 min at room temperature (RT) without breaks. The interphasecontaining the PBMCs was collected and washed three times with PBS(350×g followed by 300×g for 10 min at RT).

Subsequently, PBMCs were labeled with 40 nM CFSE (carboxyfluoresceinsuccinimidyl ester) for 15 min at 37° C. Cells were washed with 20 mlmedium before recovering the labeled PBMCs for 30 min at 37° C. Thecells were washed, counted, and 100000 cells were seeded into96-well-U-bottom plates. Pre-diluted Proleukin (commercially availablewild-type IL-2) or IL2-immunoconjugates were titrated onto the seededcells which were incubated for the indicated time points. After 4-6days, cells were washed, stained for appropriate cell surface markers,and analyzed by FACS using a BD FACSCantoII. NK cells were defined asCD37CD56⁺, CD4 T cells as CD3⁺/CD8⁻, and CD8 T cells as CD3⁺/CD8⁺.

FIGS. 33A-C show proliferation of NK cells after incubation withdifferent FAP-targeted 28H1 IL-2 immunoconjugates for 4 (FIG. 33A), 5(FIG. 33B) or 6 (FIG. 33C) days. All tested constructs induced NK cellproliferation in a concentration-dependent manner. Proleukin was moreefficacious than the immunoconjugates at lower concentrations, thisdifference no longer existed at higher concentrations, however. Atearlier time points (day 4), the IgG-IL2 constructs appeared slightlymore potent than the Fab-IL2-Fab constructs. At later time points (day6), all constructs had comparable efficacy, with the Fab-IL2 qm-Fabconstruct being least potent at the low concentrations.

FIGS. 34A-C show proliferation of CD4 T-cells after incubation withdifferent FAP-targeted 28H1 IL-2 immunoconjugates for 4 (FIG. 34A), 5(FIG. 34B) or 6 (FIG. 34C) days. All tested constructs induced CD4 Tcell proliferation in a concentration-dependent manner. Proleukin had ahigher activity than the immunoconjugates, and the immunoconjugatescomprising wild-type IL-2 were slightly more potent than the onescomprising quadruple mutant IL-2. As for the NK cells, the Fab-IL2qm-Fab construct had the lowest activity. Most likely the proliferatingCD4 T cells are partly regulatory T cells, at least for the wild-typeIL-2 constructs.

FIGS. 35A-C show proliferation of CD8 T-cells after incubation withdifferent FAP-targeted 28H1 IL-2 immunoconjugates for 4 (FIG. 35A), 5(FIG. 35B) or 6 (FIG. 35C) days. All tested constructs induced CD8 Tcell proliferation in a concentration-dependent manner. Proleukin had ahigher activity than the immunoconjugates, and the immunoconjugatescomprising wild-type IL-2 were slightly more potent than the onescomprising quadruple mutant IL-2. As for the NK and CD4 T cells, theFab-IL2 qm-Fab construct had the lowest activity.

Example 11 Proliferation and Activation Induced Cell Death of IL-2Activated PBMCs

Freshly isolated PBMCs from healthy donors were pre-activated overnightwith PHA-M at 1 μg/ml in RPMI1640 with 10% FCS and 1% Glutamine. Afterpre-activation PBMCs were harvested, labeled with 40 nM CFSE in PBS, andseeded in 96-well plates at 100 000 cells/well. Pre-activated PBMCs werestimulated with different concentrations of IL-2 immunoconjugates (4B9IgG-IL-2 wt, 4B9 IgG-IL-2 qm, 4B9 Fab-IL-2 wt-Fab, and 4B9 Fab-IL-2qm-Fab). After six days of IL-2 treatment PBMCs were treated with 0.5μg/ml activating anti-Fas antibody overnight. Proliferation of CD4(CD3⁺CD8⁻) and CD8 (CD3⁺CD8⁺) T cells was analyzed after six days byCFSE dilution. The percentage of living T cells after anti-Fas treatmentwas determined by gating on CD3⁺ Annexin V negative living cells.

As shown in FIGS. 36A-B, all constructs induced proliferation ofpre-activated T cells. At low concentrations the constructs comprisingwild-type IL-2 wt were more active than the IL-2 qm-comprisingconstructs. IgG-IL-2 wt, Fab-IL-2 wt-Fab and Proleukin had similaractivity. Fab-IL-2 qm-Fab was slightly less active than IgG-IL-2 qm. Theconstructs comprising wild-type IL-2 were more active on CD4 T cellsthan on CD8 T cells, most probably because of the activation ofregulatory T cells. The constructs comprising quadruple mutant IL-2 weresimilarly active on CD8 and CD4 T cells.

As shown in FIG. 37, T cells stimulated with high concentrations ofwild-type IL-2 are more sensitive to anti-Fas induced apoptosis than Tcells treated with quadruple mutant IL-2.

Example 12

The untargeted DP47GS construct (see SEQ ID NO: 299 and 297 for VH andVL sequences, respectively) was further characterized. As describedabove, conjugates of DP47GS IgG with wild-type or quadruple mutant IL-2were made. These constructs showed similar binding to IL-2R andinduction of immune cell (e.g. NK cell, CD8⁺ cell and CD4⁺ cell)proliferation in vitro as corresponding targeted constructs (data notshown). In contrast to immunoconjugates targeting a tumor antigen,however, they did not accumulate in tumor tissue (see Example 8).

A further pharmacokinetic study (in addition to the one shown in Example7) was performed with the untargeted DP47GS IgG-IL-2 constructscomprising either wild-type or quadruple mutant IL-2. Male C57BL/6J mice(n=6 per group) were injected i.v. with 0.3, 1, 3 or 10 mg/kg DP47GSIgG-IL-2 wt or DP47GS IgG-IL-2 qm construct. The injection volume was 1ml for all mice. Blood samples were taken at 2, 4, 8, 24, 48, 72, 96 and168 hours after injection (from 3 mice at each time point) and stored at−20° C. until analysis. The constructs were quantified in the serumsamples by ELISA, using anti-Fab antibodies for capturing and detectionof the constructs. All samples and calibration standards were diluted1:25 in mouse serum (obtained from Bioreclamation) prior to theanalysis. Briefly, streptavidin-coated 96 well plates (Roche) werewashed three times for 10 sec with PBS/0.05% Tween 20, before incubationwith 100 μl/well (0.5 μg/ml) biotinylated anti-human Fab antibody(M-1.7.10; Roche Diagnostics) for 1 hour at room temperature. Afterwashing the plate again three times with PBS/0.05% Tween 20, 50 μl/wellof the serum samples or calibration standards and 50 μl/well PBS/0.5%BSA were added to give a final sample dilution of 1:50. Samples wereincubated for 1 hour at room temperature, followed by washing the plateagain three times with PBS/0.05% Tween 20. Next, the plate was incubatedwith 100 μl/well (0.5 μg/ml) digoxigenin-labeled anti-human Fab antibody(M-1.19.31; Roche Diagnostics) for 1 hour at room temperature, washed,incubated with 100 μl/well anti-digoxigenin POD (Roche DiagnosticsCat#11633716001) for 1 hour at room temperature, and washed again.Finally, 100 μl/well TMB peroxidase substrate (Roche DiagnosticsCat#11484281001) was added for about 5 minutes, before the substratereaction was stopped with 50 μl/well 2N HCl. The plate was read within 2minutes after stopping the reaction at 450 nm with a referencewavelength of 650 nm.

The result of this study is shown in FIGS. 38A-B. Both constructs showedlong serum half life, with the construct comprising quadruple mutantIL-2 (FIG. 38B) being even longer lived than the one comprisingwild-type IL-2 (FIG. 38A).

In addition, the lack of binding of DP47GS IgG to various proteins aswell as human cells (PBMCs) was confirmed.

The binding specificity (or lack of such) of the DP47GS antibody wasassessed in an ELISA-based test system with a panel of differentunrelated antigens. The test was performed on 384 well MaxiSorp™microtiter plates (Thermo Scientific Nunc, Cat#460372). After eachincubation step the plates were washed three times with PBS/0.05%Tween-20. First, the different antigens, diluted in PBS, were coated onplates overnight at 6° C. The test concentrations and detailedinformation for the used antigens are listed in the table below.

Test con- centration Antigen Source Supplier Cat# [μg/ml] Histons calfRoche 10223565601 2 thymus Diagnostics BSA bovine Roche 10735108001 2Fraction V Diagnostics Insulin human Roche 11376497001 2 DiagnosticsCardiolipin bovine Sigma-Aldrich C1649 2 Heparin porcine Sigma-AldrichH9902 2 CD40 (hFc) human Sino Biological 1077-H03H 1 Parathyroid humanAnaSpec    20690 0.5 hormone aa 1-34 (PTH) (biotinylated) dsDNA calfthymus Sigma-Aldrich D4522 0.16 Hemocyanin keyhole Sigma-Aldrich H70170.22 limpet Actin beta 2 human Cytoskeleton APHL99 0.67 StreptavidinStreptomyces Roche 11721674001 1 avidinii Diagnostics Gelatin bovineRoche 11111965001 2% Diagnostics blocking buffer E. coli lysate E. coliinhouse — diluted 1:600

Thereafter, the wells were blocked with 2% gelatin in water for 1 hourat room temperature (RT). The DP47GS antibody (1 μg/ml in PBS) wasincubated with the panel of captured antigens for 1.5 hours at RT. Boundantibody was detected using anti-human IgG antibody-HRP conjugate (GEHealthcare, Cat#9330V; diluted 1:1000 in PBS with 0.2% Tween-20 and 0.5%gelatin). After 1 hour incubation the plates were washed 6 times withPBS/0.05% Tween-20 and developed with freshly prepared BM blue PODsubstrate solution (BM blue: 3,3′-5,5′-tetramethylbenzidine, RocheDiagnostics, Cat#11484281001) for 30 minutes at RT. Absorbance wasmeasured at 370 nm. The blank value was defined without addition ofantibody. An inhouse human IgG₁ antibody which exhibits unspecificbinding to almost all of the captured antigens served as positivecontrol.

The result of this experiment is shown in FIG. 39. The DP47GS antibodyshowed no binding to any of the captured antigens. The detected signalswere in the range of the control samples without antibody.

Finally, the binding of the DP47GS antibody to human PBMCs was assessed.Since in the course of a typical immune response the combination of cellsurface-presented proteins changes dramatically, binding was tested onPBMCs directly after isolation from healthy adults as well as after invitro activation with two different stimuli.

Human PBMCs were isolated by Ficoll density gradient centrifugation frombuffy coats or from heparinized fresh blood from healthy volunteersusing Histopaque 1077 (Sigma-Aldrich, Germany). PBMCs were eitherdirectly subjected to binding assays (fresh PBMCs) or cultured andstimulated further. PBMCs were cultured at a cell density of 2×10⁶cells/ml in T cell medium consisting of RPMI 1640 (Gibco) supplementedwith 10% (v/v) heat-inactivated FBS (PAA Laboratories), 1 mM sodiumpyruvate (Sigma-Aldrich), 1% (v/v) L-alanyl-L-gluthamine (Biochrom) and10 nM β-mercaptoethanol (Sigma-Aldrich) at 37° C. For in vitrostimulation, Proleukin (200 U/ml, Novartis) and phytohaemagglutinin(PHA-L; 2 μg/mL, Sigma-Aldrich) were added during six days ofcultivation (PHA-L activated PBMC). For in vitro re-stimulation, 6-wellcell culture plates were coated with mouse anti-human CD3 (clone KT3, 1μg/ml) and mouse anti-human CD28 antibodies (clone 28.2, 2 μg/ml, bothfrom eBioscience) and PHA-L activated PBMC were added for additional 24hours (re-stimulated PBMC). Binding of DP47GS antibody (with or withoutthe L234A L235A (LALA) P329G mutation in the Fc domain) to cell surfaceproteins was monitored for a five-fold serial dilution series (highestconcentration 200 nM) using a goat anti-human IgG Fc-specific secondaryantibody conjugated to fluorescein isothiocyanate (FITC) (JacksonLaboratories) and flow cytometric analysis. All assays were performed at4° C. to prevent internalization of surface proteins. Incubation ofprimary and secondary antibody was for 2 hours and for 1 hour,respectively. To allow simultaneous typing of leukocytes, combinationsof fluorochrome-labeled mouse anti-human CD14, CD15, CD4, CD19 (allBiolegend), NKp46, CD3, CD56, CD8 (all BD Pharmingen) were added to thesecondary antibody. Propidium iodide (1 μg/ml) was added directly beforemeasurement on a FACSCantoII device running FACS Diva software (both BDBioscience) to exclude permeable dead cells. Propidium iodide negativeliving cells were gated for T cells (CD14⁻CD3⁺CD4⁺/CD8⁺), B cells(CD14⁻CD19⁺), NK Cells (CD14⁻NKp46⁺/CD56⁺) or monocytes/neutrophils(CD3⁻CD56⁻CD14⁺/CD15⁺). The median FITC fluorescence of the variousleukocyte types was determined as indicator for bound primary antibodyand blotted against the primary antibody concentration using Prism4(GraphPad Software).

As shown in FIGS. 40A-L, the DP47GS IgG antibody without Fc mutationshowed binding only to Fcγ receptor bearing cells, e.g. NK cells andmonocytes/neutrophils. No binding of DP47GS (LALA P329G) was detected onhuman PBMCs, regardless of their activation status. The LALA P329Gmutation in the Fc domain completely abolished binding also to Fcγreceptor bearing cells.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

1. An immunoconjugate comprising (i) an immunoglobulin molecule comprising a first and a second antigen binding Fab molecule, an Fc domain consisting of two subunits, and (ii) an effector moiety, wherein not more than one effector moiety is present; and wherein said first and said second Fab molecule are directed to CEA and comprise a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 191, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 189; and said effector moiety is a mutant human interleukin-2 (IL-2) polypeptide comprising the amino acid substitutions F42A, Y45A, L72G, wherein numbering of the amino acid positions is relative to the human IL-2 sequence in SEQ ID NO:
 2. 2. The immunoconjugate of claim 1, wherein said effector moiety is fused to the carboxy-terminal amino acid of one of said two subunits of the Fc domain, optionally through a linker peptide.
 3. The immunoconjugate of claim 1, wherein said Fc domain comprises a modification promoting heterodimerization of two non-identical polypeptide chains.
 4. The immunoconjugate of claim 3, wherein said modification is a knob-into-hole modification, comprising a knob modification in one of the subunits of the Fc domain and a hole modification in the other one of the two subunits of the Fc domain.
 5. The immunoconjugate of claim 4, wherein said knob modification comprises the amino acid substitution T366W, and said hole modification comprises the amino acid substitutions T366S, L368A and Y407V according to the EU numbering of Kabat.
 6. The immunoconjugate of claim 5, wherein the subunit of the Fc domain comprising the knob modification additionally comprises the amino acid substitution S354C, and the subunit of the Fc domain comprising the hole modification additionally comprises the amino acid substitution Y349C according to the EU numbering of Kabat.
 7. The immunoconjugate of claim 4, wherein said effector moiety is fused to the carboxy-terminal amino acid of the subunit of the Fc domain comprising the knob modification.
 8. The immunoconjugate of claim 1, wherein said Fc domain is an IgG Fc domain.
 9. The immunoconjugate of claim 1, wherein said Fc domain is an IgG1 Fc domain.
 10. The immunoconjugate of claim 1, wherein said Fc domain is engineered to have altered binding to an Fc receptor and/or altered effector function.
 11. The immunoconjugate of claim 10, wherein said Fc receptor is an Fcγ receptor.
 12. The immunoconjugate of claim 10, wherein said effector function is ADCC.
 13. The immunoconjugate of claim 10, wherein said altered binding and/or effector function is reduced binding and/or effector function.
 14. The immunoconjugate of claim 13, wherein said Fc domain comprises one or more amino acid mutation that reduces the binding of the Fc domain to an Fc receptor, particularly an Fcγ receptor.
 15. The immunoconjugate of claim 14, wherein said amino acid mutation is an amino acid substitution at position P329 according to the EU numbering of Kabat.
 16. The immunoconjugate of claim 14, wherein the Fc domain comprises the amino acid substitutions L234A, L235A and P329G according to the EU numbering of Kabat in each of said two subunits.
 17. The immunoconjugate of claim 10, wherein said altered binding and/or effector function is increased binding and/or effector function.
 18. The immunoconjugate of claim 17, wherein said Fc domain is engineered to have an altered oligosaccharide structure, as compared to a non-engineered Fc domain.
 19. The immunoconjugate of claim 18, wherein said Fc domain comprises an increased proportion of non-fucosylated oligosaccharides, as compared to a non-engineered Fc domain.
 20. The immunoconjugate of claim 1, wherein said immunoglobulin molecule is an IgG class immunoglobulin.
 21. The immunoconjugate of claim 1, wherein said immunoglobulin molecule is an IgG1 class immunoglobulin.
 22. The immunoconjugate of claim 1, wherein said effector moiety is fused to the carboxy-terminal amino acid of one of the immunoglobulin heavy chains, optionally through a linker peptide.
 23. The immunoconjugate of claim 1, wherein said mutant human interleukin-2 (IL-2) polypeptide further comprises the amino acid substitution T3A.
 24. The immunoconjugate of claim 1, wherein said mutant human interleukin-2 (IL-2) polypeptide further comprises the amino acid substitution C125A.
 25. The immunoconjugate of claim 1, wherein said mutant human interleukin-2 (IL-2) polypeptide comprises the polypeptide sequence of SEQ ID NO:
 3. 26. The immunoconjugate of claim 1, comprising the polypeptide sequences of SEQ ID NO: 277, SEQ ID NO: 281 and SEQ ID NO:
 283. 27. An isolated polynucleotide encoding the immunoconjugate of claim 1 or a fragment thereof.
 28. An expression vector comprising the isolated polynucleotide of claim
 27. 29. A host cell comprising the expression vector of claim
 28. 30. A method of producing an immunoconjugate comprising (i) an immunoglobulin molecule comprising a first and a second antigen binding Fab molecule and an Fc domain consisting of two subunits, and (ii) an effector moiety, wherein not more than one effector moiety is present, comprising culturing the host cell of claim 29 under conditions suitable for the expression of the immunoconjugate and optionally recovering the immunoconjugate.
 31. An immunoconjugate comprising (i) an immunoglobulin molecule comprising a first and a second antigen binding Fab molecule and an Fc domain consisting of two subunits, and (ii) an effector moiety, wherein not more than one effector moiety is present, wherein said immunoconjugate is produced by the method of claim
 30. 32. A pharmaceutical composition comprising the immunoconjugate of claim 26 and a pharmaceutically acceptable carrier.
 33. A method of treating a disease in an individual, comprising administering to said individual a therapeutically effective amount of a composition comprising the immunoconjugate of claim 26 in a pharmaceutically acceptable form.
 34. The method of claim 33, wherein said disease is cancer.
 35. The method of claim 33, wherein said disease is an inflammatory disorder. 